EA047139B1 - COMPOSITIONS - Google Patents

Description

The invention claims benefit from U.S. Provisional Patent Application No. 62/566,240, filed September 29, 2017, the contents of which are incorporated herein by reference in their entirety.

This document proposes compositions of lipid nanoparticles (LNPs) with improved properties for the delivery of biologically active agents, in particular RNA, mRNA and guide RNAs. LNP compositions facilitate the delivery of RNA agents across cell membranes, and in particular embodiments, they introduce gene editing components and compositions into living cells.

Biologically active agents that are particularly difficult to deliver into cells include proteins, nucleic acid drugs, and their derivatives. Of particular interest are compositions for delivering promising gene editing technologies into cells, for example, for delivering components of the CRISPR/Cas9 system.

There are now a number of components and systems for gene editing in cells in vivo, providing enormous potential for treating diseases. CRISPR/Cas gene editing systems are active in cells in the form of ribonucleoprotein complexes. In a cell, an RNA-directed nuclease binds to a DNA sequence and directs its cleavage. This site-specific nuclease activity facilitates gene editing through the cell's own natural processes. For example, the cell responds to DNA double-strand breaks (DSBs) with an error-prone repair process known as non-homologous end joining (NHEJ). During NHEJ, nucleotides can be added or removed by the cell from the ends of the DNA, resulting in a sequence change from the cleaved sequence. In other cases, cells repair DSBs using homology-directed repair (HDR) or homologous recombination (HR) mechanisms, in which an endogenous or exogenous template can be used to directly repair the break. Some of these editing technologies use cellular machinery to repair single-strand breaks (SSBs) or DSBs.

There is a need for compositions for delivering CRISPR/Cas protein and nucleic acid components into a cell, such as a patient cell. In particular, compositions for delivering mRNA encoding the protein component of CRISPR and for delivering CRISPR guide RNAs are of particular interest. Compositions with useful in vitro and in vivo delivery properties that can stabilize and deliver RNA components are also of particular interest.

Herein, we provide lipid nanoparticle compositions with useful properties, particularly for the delivery of CRISPR/Cas gene editing components.

In certain embodiments, the LNP compositions contain: an RNA component and a lipid component, wherein the lipid component contains: (1) about 50-60 mol% aminolipid; (2) about 8-10 mol% neutral lipid; and (3) about 2.5-4 mol% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, and wherein the N/P ratio of the LNP composition is about 6. In further embodiments, the LNP compositions comprise (1) RNA component; (2) about 50-60 mol% aminolipid; (3) about 27-39.5 mol% auxiliary lipid; (4) about 8-10 mol% neutral lipid; and (5) about 2.5-4 mol% PEG lipid, wherein the N/P ratio of the LNP composition is about 5-7.

In other embodiments, LNP compositions contain an RNA component and a lipid component, wherein the lipid component contains: (1) about 50-60 mol% aminolipid; (2) about 5-15 mol% neutral lipid; and (3) about 2.5-4 mol% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, and wherein the N/P ratio of the LNP composition is about 3-10. In additional embodiments, the LNP compositions contain a lipid component that contains (1) about 40-60 mol% aminolipid; (2) about 5-15 mol% neutral lipid; and (3) about 2.5-4 mol.% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, and wherein the N/P ratio of the LNP composition is about 6. In another embodiment, the LNP compositions contain a lipid component that contains (1) about 50-60 mol% aminolipid; (2) about 5-15 mol% neutral lipid; and (3) about 1.5-10 mol% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, and wherein the N/P ratio of the LNP composition is about 6.

In some embodiments, the LNP compositions contain an RNA component and a lipid component, wherein the lipid component contains: (1) about 40-60 mol% aminolipid; (2) about 0-5 mol% neutral lipid, such as a phospholipid; and (3) about 1.5-10 mol% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, and wherein the N/P ratio of the LNP composition is about 3-10. In some embodiments, the LNP compositions contain an RNA component and a lipid component, wherein the lipid component contains: (1) about 40-60 mol% aminolipid; (2) less than about 1 mol% neutral lipid, such as a phospholipid; and (3) about 1.5-10 mol% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, and wherein the N/P ratio of the LNP composition is about 3-10. In certain embodiments, the LNP composition is substantially free of neutral lipid. In some embodiments, the LNP compositions contain an RNA component and a lipid component, wherein the lipid component is

- 1 047139 contains: (1) about 40-60 mol.% aminolipid; and (2) about 1.5-10 mol% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, wherein the N/P ratio of the LNP composition is about 3-10, and where the LNP composition does not contain a neutral lipid, e.g. phospholipid. In certain embodiments, the LNP composition contains substantially no or no neutral phospholipid. In certain embodiments, the LNP composition contains substantially no or no neutral lipid, such as a phospholipid.

In certain embodiments, the RNA component comprises mRNA, such as an RNA-guided DNA binding agent (eg, a Cas nuclease or class 2 Cas nuclease). In certain embodiments, the RNA component comprises gRNA.

Brief description of graphic materials

In fig. Figure 1 shows the percentage of TTR gene editing achieved in mouse liver following delivery of CRISPR/Cas gene editing components, Cas9 mRNA and gRNA, in LNP formulations as indicated by a single dose of 1 mg/kg body weight (Figure 1A) or 0. 5 mg/kg body weight (Fig. 1B).

In fig. Figure 2 shows particle distribution data for LNP compositions containing Cas9 mRNA and gRNA.

In fig. Figure 3 shows the physicochemical properties of the LNP compositions by comparing the log derived molar masses (Figure 3A) and the average molecular weight values (Figure 3B) for these compositions.

In fig. Figure 4 shows polydispersity calculations (Figure 4A) and a Burchard-Stockmeier analysis (Figure 4B) that analyzes the LNP compositions from Figure 4. 3.

In fig. Figure 5 presents the results of an experiment evaluating the effects of LNP formulations with increased concentrations of PEG lipids on serum TTR knockdown, hepatic gene editing, and MCP-1 cytokine levels after a single dose in rats. In fig. 5A shows a graph of serum TTR levels; in fig. 5B shows a graph of percent editing in liver samples; and in fig. Figure 5C shows MCP-1 levels in pg/ml.

In fig. Figure 6 shows that LNP formulations retain gene editing ability with various PEG lipids (as measured by serum TTR levels (Figures 6A and 6B) and percent editing (Figure 6C).

In fig. 7 shows that Lipid A analogues effectively deliver gene editing cargo in LNP formulations, as measured by % editing in the liver after a single dose in mice.

In fig. 8 shows a dose response curve in percent editing for various LNP compositions in primary cynomolgus monkey hepatocytes.

In fig. 9A and 9B show serum TTR assay results and percentage editing when the gRNA to mRNA ratio is changed, and FIG. 9C and FIG. Figure 9D shows serum TTR assay results and percentage editing in liver when the amount of Cas9 mRNA is kept constant and gRNA is varied after a single dose in a mouse.

In fig. 10A and 10B show the results of serum TTR assay and liver editing following administration of LNP compositions with and without neutral lipid.

Detailed description of the invention

This disclosure provides embodiments of lipid nanoparticle (LNP)-based RNA compositions including CRISPR/Cas (cargo) RNA components for delivery into cells, and methods of using them. LNP compositions may exhibit improved properties compared to prior delivery technologies. The LNP composition may contain an RNA component and a lipid component, as defined herein. In certain embodiments, the RNA component includes a Cas nuclease, such as a class 2 Cas nuclease. In certain embodiments, the cargo or RNA component includes an mRNA encoding a class 2 Cas nuclease and a guide RNA or nucleic acid , encoding guide RNAs. Methods for gene editing and methods for creating engineered cells have also been proposed.

Cargo CRISPR/Cas.

CRISPR/Cas cargo delivered via an LNP formulation may contain an mRNA molecule encoding a protein of interest. For example, the cargo includes mRNA to express a protein, such as green fluorescent protein (GFP), and an RNA-guided DNA-binding agent, or Cas nuclease. LNP compositions have been proposed that contain Cas nuclease mRNA, for example class 2 Cas nuclease mRNA, which ensures the expression of the Cas9 protein in the cell. In addition, the cargo may contain one or more guide RNAs or nucleic acids encoding guide RNAs. A template nucleic acid, for example for repair or recombination, may also be included in the composition, or the template nucleic acid may be used in the methods described herein.

The term mRNA refers to a polynucleotide that contains an open reading frame that can be translated into a polypeptide (that is, it can serve as a substrate for translation by a ribosome and an aminoacylated tRNA). The mRNA may contain a sugar phosphate backbone including ribose residues or analogues thereof, such as 2'-methoxyribose residues. In some variants

- 2 047139 implementations of the sugars of the sugar phosphate backbone of mRNA consist essentially of ribose residues, 2'-methoxyribose residues, or a combination thereof. Typically, mRNAs do not contain significant amounts of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10, 9, 8, 7, 6, 5, 4 thymidine residues , 4, 3, 2, 1, 0.5, 0.2 or 0.1%). mRNA may contain modified uridines at some or all of its uridine positions.

Nuclease system CRISPR/Cas.

One component of the disclosed compositions is an mRNA encoding an RNA-guided DNA binding agent, such as a Cas nuclease.

As used herein, the term RNA-directed DNA-binding agent means a polypeptide or complex of polypeptides having RNA-DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA-binding activity is sequence specific and dependent on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas clevases/nickases and inactivated forms thereof (dCas DNA binding agents). The term Cas nuclease as used herein covers Cas clevases, Cas nicases and dCas DNA binding agents. Cas clevases/nickases and dCas DNA binding agents include the Csm or Cmr complex of the type III CRISPR system, its subunit Cas10, Csm1 or Cmr2, the Cascade complex of the type I CRISPR system, its subunit Cas3 and class 2 Cas nucleases. Used in As used herein, the term Cas nuclease class 2 is a single-stranded polypeptide with RNA-directed DNA-binding activity. Class 2 Cas nucleases include class 2 Casclevases/nickases (e.g., H840A, D10A, or N863A variants), which additionally have RNA-guided clevase or nickase activity on DNA, and class 2 dCas DNA-binding agents, in which the clease activity is nickase is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (for example, variants N497A, R661A, Q695A, Q926A), HypaCas9 (for example, variants N692A, M694A, Q695A, H698A), eSPCas9 ( 1.0) (for example, variants K81OA, K1OOZA, R1060A) and eSPCas9(1.1) (for example, variants K848A, K1OOZA, R1060A) and their modifications. The Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9 and contains a RuvC-like nuclease domain. Cpf1 sequences from Zetsche are incorporated by reference in their entirety. See, for example, Zetsche, tables S1 and S3. See, for example, Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

In some embodiments, the RNA-guided DNA binding agent is a class 2 nuclease. In some embodiments, the RNA-guided DNA binding agent has clease activity, which may also be referred to as double-stranded endonuclease activity. In some embodiments, the RNA-guided DNA binding agent comprises a Cas nuclease, such as a class 2 Cas nuclease (which may be, for example, a type II, V, or VI Cas nuclease). Class 2 Cas nucleases include, for example, the Cas9, Cpf1, C2c1, C2c2 and C2c3 proteins, and modifications thereof. Examples of Cas9 nucleases include the type II CRISPR systems of S. pyogenes, S. aureus and other prokaryotes (see, for example, the list in the next paragraph) and their modified (eg, engineered or mutant) variants. See, for example, U.S. 2016/0312198 A1; U.S. 2016/0312199 A1. Other examples of Cas nucleases include the Csm or Cmr complex of the type III CRISPR system or its subunit Cas10, Csm1 or Cmr2; and the Cascade complex of the type I CRISPR system or its subunit Cas3. In some embodiments, the Cas nuclease may be from a type IIA, type IIB, or type IIC system. For a discussion of various CRISPR systems and Cas nucleases, see, for example, Makarova et al., Nat. Rev. Microbiol. 9:467-477 (2011); Makarova et al., Nat. Rev. Microbiol, 13: 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

Non-limiting illustrative species from which the Cas nuclease may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus se lenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola , Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum , Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, ularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae sp. ND2006, and Acaryochloris marina.

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In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus thermophilus. In some embodiments, the Cas nuclease is a Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is a Cas9 nuclease from Staphylococcus aureus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella novicida. In some embodiments, the Casnuclease is a Cpf1 nuclease from Acidaminococcus sp. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Lachnospiraceae sp ND2006. In other embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella tularensis, Lachnospiraceae sp., Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas creviorican is, Prevotella disiens or Porphyromonas macacae. In certain embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus or Lachnospiraceae.

Wild-type Cas9 has two nuclease domains, RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target DNA strand. In some embodiments, the Cas9 nuclease contains more than one RuvC domain and/or more than one HNH domain. In some embodiments, the Cas9 nuclease is wild-type Cas9. In some embodiments, Cas9 is capable of inducing a double-strand break in target DNA. In certain embodiments, the Cas nuclease may cleave dsDNA, it may cleave a single strand of dsDNA, or it may have no clease or nickase activity on DNA. An exemplary Cas9 amino acid sequence is provided as SEQ ID NO: 3. An exemplary Cas9 mRNA ORF sequence that contains start and stop codons is provided as SEQ ID NO: 4. An exemplary Cas9 mRNA coding sequence suitable for inclusion in a fusion protein is presented as SEQ ID NO: 10.

In some embodiments, chimeric Cas nucleases are used where one domain or region of a protein is replaced with part of another protein. In some embodiments, the Casnuclease domain may be replaced with a domain from another nuclease, such as Fok1. In some embodiments, the Cas nuclease may be a modified nuclease.

In other embodiments, the Cas nuclease may be derived from the Type I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of the Type I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be derived from a type III CRISPR/Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

In some embodiments, the RNA-guided DNA binding agent has single-stranded nickase activity, that is, it can cut one strand of DNA to form a single-strand break, also known as a single-strand break. In some embodiments, the RNA-guided DNA binding agent comprises a Cas nickase. Nikase is an enzyme that creates a single-strand break in dsDNA, that is, it cuts one strand but not the other strand of the DNA double helix. In some embodiments, a Cas nickase is a variant of a Casnuclease (eg, the Cas nuclease described above) in which the endonucleolytic active site is inactivated, for example, by one or more changes (eg, point mutations) in the catalytic domain. See, for example, US Pat. No. 8,889,356 for a discussion of Cas nickases and illustrative variations of the catalytic domain. In some embodiments, a Cas nickase, such as Cas9 nickase, has an inactivated RuvC or HNH domain. An exemplary amino acid sequence of Cas9 nickase is given as SEQ ID NO: 6.

An exemplary Cas9 nickase mRNA ORF sequence that contains start and stop codons is shown as SEQ ID NO: 7. An exemplary Cas9 nickase mRNA coding sequence suitable for inclusion in a fusion protein is shown as SEQ ID NO: 11.

In some embodiments, the RNA-guided DNA binding agent is modified so that it contains only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated, either completely or partially, to reduce its nucleic acid cleaving activity. In some embodiments, a nickase having a RuvC domain with reduced activity is used. In some embodiments, a nickase having an inactive RuvC domain is used. In some embodiments, a nickase having a reduced activity HNH domain is used. In some embodiments, a nickase having an inactive HNH domain is used.

In some embodiments, a conserved amino acid in the Cas nuclease protein domain is replaced to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, for example, Zetsche et al. (2015) Cell Oct 22:163(3):

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759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863 A, H983 A, and D986A (based on the S. pyogenes Cas9 protein). See, for example, Zetsche et al. (2015). Other exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB - A0Q7Q2 (CPF1_FRATN)).

In some embodiments, the mRNA encoding the nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, guide RNAs direct nickase to the target sequence and introduce DSBs by creating single-strand breaks on opposite strands of the target sequence (ie, a double single-strand break). In some embodiments, the use of a double single-strand break may improve specificity and reduce side effects. In some embodiments, nickase is used in conjunction with two separate guide RNAs targeting opposing DNA strands to create a double single-strand break in the target DNA. In some embodiments, nickase is used in conjunction with two separate guide RNAs that are selected to be in close proximity to create a double single-strand break in the target DNA.

In some embodiments, the RNA-guided DNA binding agent does not have clease or nickase activity. In some embodiments, the RNA-guided DNA binding agent comprises a dCas DNA binding polypeptide. The dCas polypeptide has DNA-binding activity but essentially no catalytic (clevase/nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-binding agent that does not have clease or nickase activity, or the dCas DNA-binding polypeptide, is a variant of a Cas nuclease (e.g., the Cas nuclease described above) in which its endonucleolytic active sites are inactivated, e.g. by one or more changes (eg, point mutations) in its catalytic domains. See, for example, U.S. 2014/0186958 A1; U.S. 2015/0166980 A1. An exemplary dCas9 amino acid sequence is provided as SEQ ID NO: 8. An exemplary Cas9 mRNA ORF sequence that contains start and stop codons is provided as SEQ ID NO: 9. An exemplary Cas9 mRNA coding sequence suitable for inclusion in a fusion protein is presented as SEQ ID NO: 12.

In some embodiments, the RNA-guided DNA binding agent comprises one or more heterologous functional domains (eg, is or contains a fusion polypeptide).

In some embodiments, the heterologous functional domain may facilitate the transport of an RNA-guided DNA binding agent into the cell nucleus. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA binding agent may be fused to 1-10 NLSs. In some embodiments, the RNA-guided DNA binding agent may be fused to the 15 NLS. In some embodiments, the RNA-guided DNA binding agent may be fused to a single NLS. If a single NLS is used, the NLS may be linked to the N-terminus or C-terminus of the RNA-guided DNA binding agent sequence. It can also be inserted into an RNA-guided DNA binding agent sequence. In other embodiments, the RNA-guided DNA binding agent may be fused to more than one NLS. In some embodiments, the RNA-guided DNA binding agent may be fused to 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guided DNA binding agent may be fused to two NLSs. In certain circumstances, two NLSs may be the same (for example, two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA binding agent is fused to two SV40 NLS sequences linked at the C terminus. In some embodiments, the RNA-guided DNA binding agent may be fused to two NLSs, one linked at the N-terminus and the other linked at the C-terminus. In some embodiments, the RNA-guided DNA binding agent may be fused to the 3 NLS. In some embodiments, the RNA-directed DNA binding agent may be fused without an NLS. In some embodiments, the NLS may be a single sequence, such as, for example, SV40 NLS, PKKKRKV, or PKKKRRV. In some embodiments, the NLS may be a double sequence, such as the nucleoplasmin NLS, KRPAATKKAGQAKKKK. In a specific embodiment, one PKKKRKV NLS may be linked at the C-terminus of the RNA-guided DNA binding agent. One or more linkers are optionally included at the fusion site.

In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of an RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided

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DNA binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA binding agent may be modified by the addition of ubiquitin or a polyubiquitin chain. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), protein 8, suppresses the expression of a set of genes in neural precursors during development (NEDD8, also called Rub1 in S. cerevisiae), human leukocyte F antigen-associated protein (FAT10), autophagy protein-8 (ATG8) and -12 (ATG12), ubiquitin-like Fau protein (FUB1), membrane-anchored UBL (MUB), ubiquitin folded modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).

In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g. EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), blue fluorescent proteins (e.g. ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g. mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mStrawberry, Jred) and orange fluorescent proteins (mOrange, mKO, KusabiraOrange, monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and/or an epitope tag. Non-limiting exemplary labels include glutathione Stransferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), no.in (NANP). tag for tandem affinity purification (TAP), myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1 , T7, V5, VSV-G, 6xHis, 8xHis, biotin carboxyl carrier protein (BCCP), poly-His and calmodulin. Non-limiting exemplary reporter genes include glutathione-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.

In additional embodiments, the heterologous functional domain can target the RNA-guided DNA binding agent to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain can target the RNA-guided DNA binding agent to mitochondria.

In additional embodiments, the heterologous functional domain may be an effector domain. If an RNA-targeted DNA binding agent is directed to its target sequence, for example, if a Cas nuclease is directed to the target sequence by a gRNA, the effector domain can modify or influence the target sequence. In some embodiments, the effector domain may be selected from a nucleic acid binding domain, a nuclease domain (eg, a non-Cas nuclease domain), an epigenetic modification domain, a transcription activating domain, or a transcriptional repressor domain. In some embodiments, the heterologous functional domain is a nuclease, such as FokI nuclease. See, for example, US patent no. 9023649. In some embodiments, the heterologous functional domain is a transcriptional activator or repressor. See, for example, Qi et al., Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression, Cell 152:1173-83 (2013); Perez-Pinera et al., RNA-guided gene activation by CRISPR-Cas9-based transcription factors, Nat. Methods 10:973-6 (2013); Mali et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering, Nat. Biotechnol. 31:833-8 (2013); Gilbert et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes, Cell 154:442-51 (2013). As such, an RNA-directed DNA binding agent essentially becomes a transcription factor that can be directed to bind

- 6 047139 desired target sequence using guide RNA. In certain embodiments, the DNA modification domain is a methylation domain, such as a demethylation or methyltransferase domain. In certain embodiments, the effector domain is a DNA modification domain, such as a base editing domain. In specific embodiments, a DNA modification domain is a nucleic acid editing domain that introduces a specific modification to DNA, such as a deaminase domain. See for example WO 2015/089406; US. 2016/0304846. The nucleic acid editing domains, deaminase domains and Cas9 variants described in WO 2015/089406 and US 2016/0304846 are incorporated herein by reference.

The nuclease may contain at least one domain that interacts with a guide RNA (gRNA). In addition, the nuclease can be directed to a target sequence using gRNA. In class 2 Cas nuclease systems, the gRNA interacts with the nuclease as well as the target sequence so that it directs binding to the target sequence. In some embodiments, the gRNA provides specificity for target cleavage, and the nuclease can be versatile and paired with different gRNAs to cleave different target sequences. The class 2 Cas nuclease can bind to the gRNA backbone of the types, orthologs, and exemplary species listed above.

Guide RNA (gRNA).

In some embodiments of this disclosure, the cargo for the LNP formulation comprises at least one gRNA. The gRNA can direct a Cas nuclease or class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, the gRNA binds to and provides cleavage specificity by a class 2 Cas nuclease. In some embodiments, the gRNA and the Cas nuclease can form a ribonucleoprotein (RNP), such as a CRISPR/Cas complex, such as a CRISPR/Cas9 complex, which can be delivered by the LNP composition. In some embodiments, the CRISPR/Cas complex may be a type II CRISPR/Cas9 complex. In some embodiments, the CRISPR/Cas complex may be a type V CRISPR/Cas complex, such as a Cpf1/guide RNA complex. Cas nucleases and related gRNAs can be paired. The gRNA scaffold structures that bind to each class 2 Cas nuclease vary depending on the specific CRISPR/Cas system.

The terms guide RNA, gRNA, and guide are used interchangeably herein to refer to either sgRNA (also known as CRISPR RNA) or a combination of sgRNA and trRNA (also known as tracrRNA). sgRNA and trRNA can be linked as a single RNA molecule (single guide RNA, sgRNA) or as two separate RNA molecules (double guide RNA, dgRNA). The term guide RNA or gRNA refers to each type. The trRNA may be a naturally occurring sequence or a trRNA sequence with modifications or variations compared to naturally occurring sequences.

As used herein, the term guide sequence refers to a sequence in a guide RNA that is complementary to a target sequence and functions to direct the guide RNA to the target sequence for binding or modification (eg, cleavage) by an RNA-guided DNA binding agent. A guide sequence may also be referred to as a targeting sequence or spacer sequence. The guide sequence may be 20 base pairs in length, for example in the case of Streptococcus pyogenes (ie Spy Cas9) and related Cas9 homologs/orthologues. Shorter or longer sequences can also be used as guides, for example 15, 16, 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length. In some embodiments, the target sequence, for example, is located in a gene or chromosome and is complementary to a guide sequence. In some embodiments, the degree of complementarity or identity between the guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3 or 4 mismatches, with the total length of the target sequence being at least 17, 18, 19, 20 or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches where the guide sequence contains at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches when the guide sequence contains 20 nucleotides.

Target sequences for Cas proteins include both the positive and negative strands of genomic DNA (ie, the target sequence and the reverse complement of the sequence) because the nucleic acid substrate for the Cas protein is a double-stranded nucleic acid. Accordingly, when they say that the guide sequence is a complete

- 7 047139 mental target sequence, it should be understood that the guide sequence can direct the guide RNA to bind to the reverse complement of the target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the target sequence is identical to some nucleotides of the target sequence (eg, a non-PAM target sequence) except for the substitution of U for T in the guide sequence.

The length of the target sequence may depend on the CRISPR/Cas system and the components used. For example, different class 2 Cas nucleases from different bacterial species have different optimal lengths of targeting sequences. Accordingly, the targeting sequence may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 nucleotides in length. In some embodiments, the length of the targeting sequence is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence from the naturally occurring CRISPR/Cas system. In certain embodiments, the Cas nuclease and gRNA backbone will be derived from the same CRISPR/Cas system. In some embodiments, the targeting sequence may contain or consist of 18-24 nucleotides. In some embodiments, the targeting sequence may contain or consist of 19-21 nucleotides. In some embodiments, the targeting sequence may contain or consist of 20 nucleotides.

In some embodiments, the ssRNA is a Cas9 ssRNA capable of mediating RNA-guided DNA cleavage using a Cas9 protein. In some embodiments, the ssRNA is a Cpf1 ssRNA capable of mediating RNA-directed DNA cleavage by the Cpf1 protein. In certain embodiments, the gRNA comprises a sgRNA and a tracrRNA sufficient to form an active complex with the Cas9 protein and mediate RNA-directed DNA cleavage. In certain embodiments, the gRNA comprises a sgRNA sufficient to form an active complex with the Cpf1 protein and mediate RNA-directed DNA cleavage. See Zetsche 2015.

Certain embodiments of the invention also provide nucleic acids, such as expression cassettes, encoding a gRNA described herein. Guide RNA nucleic acid is used herein to refer to a guide RNA (eg, ssRNA or dgRNA) and a guide RNA expression cassette, which is a nucleic acid that encodes one or more guide RNAs.

In some embodiments, the nucleic acid may be a DNA molecule. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding an sgRNA. In some embodiments, the nucleotide sequence encoding the sgRNA comprises a targeting sequence flanked by all or part of a repetitive sequence from a naturally occurring CRISPR/Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding tracrRNA. In some embodiments, sgRNA and tracrRNA may be encoded by two separate nucleic acids. In other embodiments, sgRNA and tracrRNA may be encoded by the same nucleic acid. In some embodiments, sgRNA and tracrRNA may be encoded by opposite strands of the same nucleic acid. In other embodiments, sgRNA and tracrRNA may be encoded by the same strand of a single nucleic acid. In some embodiments, the gRNA nucleic acid encodes an sRNA. In some embodiments, the gRNA nucleic acid encodes a Cas9 nuclease gRNA. In some embodiments, the gRNA nucleic acid encodes the gRNA nuclease Cpf1.

The nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or regulatory control sequence, such as a promoter, 3' UTR, or 5' UTR. In one example, the promoter may be a tRNA promoter, such as a ^Lys3 mRNA or a tRNA chimera. See Mefferd et al., RNA 2015 21:1683-9; Scherer et al., Nucleic Acids Res. 2007 35: 2620-2628. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters also include the U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to the mouse or human U6 promoter. In some embodiments, the gRNA nucleic acid is a modified nucleic acid. In certain embodiments, the gRNA nucleic acid comprises a modified nucleoside or nucleotide. In some embodiments, the gRNA nucleic acid contains a 5'-terminal modification, such as a modified nucleoside or nucleotide, to stabilize and prevent integration of the nucleic acid. In some embodiments, the gRNA nucleic acid comprises double-stranded DNA having a 5'-terminal modification on each strand. In certain embodiments, the gRNA nucleic acid contains as a 5'-terminal modification an inverted dideoxy-T or an inverted nucleobase-deleted nucleoside or nucleotide. In some embodiments, the nucleic acid

- 8 047139 gRNA contains a tag such as biotin, desthiobiotene-TEG, digoxigenin and fluorescent markers including, for example, FAM, ROX, TAMRA and AlexaFluor.

In certain embodiments, more than one gRNA nucleic acid, such as gRNA, may be used with the CRISPR/Cas nuclease system.

Each gRNA nucleic acid may contain different targeting sequences, so that the CRISPR/Cas system cleaves more than one target sequence. In some embodiments, one or more gRNAs may have the same or different properties, such as activity or stability in the CRISPR/Cas complex. If more than one gRNA is used, each gRNA may be encoded by the same or different gRNA nucleic acids. The promoters used to drive the expression of more than one gRNA may be the same or different.

Modified RNAs.

In certain embodiments, the LNP compositions contain modified RNAs.

Modified nucleosides or nucleotides may be present in RNA, such as gRNA or mRNA. A gRNA or mRNA containing, for example, one or more modified nucleosides or nucleotides is called modified RNA to describe the presence of one or more non-naturally occurring and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A residues. G, C, and U. In some embodiments, the modified RNA is synthesized with a non-canonical nucleoside or nucleotide, referred to herein as modified.

Modified nucleosides and nucleotides may include one or more of the following: (i) a change, such as replacing one or both of the non-binding phosphate oxygens and/or one or more bonding phosphate oxygens in the backbone phosphodiester linkage (illustrative backbone modification); (ii) a change, such as replacing a ribose sugar component, such as a 2'hydroxyl on a ribose sugar (exemplary sugar modification); (iii) complete replacement of the phosphate moiety with dephospho linkers (illustrative backbone modification); (iv) modification or replacement of a naturally occurring nucleic acid base, including a non-canonical nucleic acid base (illustrative base modification); (v) replacing or modifying the ribose phosphate backbone (exemplary backbone modification); (vi) modification of the 3' end or 5' end of the oligonucleotide, such as removal, modification or replacement of a terminal phosphate group or conjugation of a fragment, cap or linker (such modifications of the 3' or 5' cap may include modification of a sugar and/or or skeleton); and (vii) modifying or replacing sugar (illustrative sugar modification). Some embodiments comprise modification of the 5' end of the mRNA, gRNA or nucleic acid. Some embodiments comprise modification of the 3' end of an mRNA, gRNA, or nucleic acid. The modified RNA may contain modifications at the 5' end and 3' end. The modified RNA may contain one or more modified residues at non-terminal positions. In certain embodiments, the gRNA contains at least one modified residue. In certain embodiments, the mRNA contains at least one modified residue.

As used herein, a first sequence is considered to contain a sequence with at least X% identity to a second sequence if an alignment of the first sequence with a second sequence demonstrates that X% or more of the positions in the second sequence are exactly the same as the first sequence. For example, the sequence AAGA contains a sequence with 100% identity to the sequence AAG, since the alignment will produce 100% identity in the sense that there are matches for all three positions of the second sequence. Differences between RNA and DNA (usually the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogues such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides since the corresponding nucleotides (such as thymidine, uridine or modified uridine) have the same same complement (eg, adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which contain guanosine or modified guanosine as a complementary base). Thus, for example, the sequence 5'-AXG, where X is any modified uridine such as pseudouridine, N1-methylpseudouridine or 5-methoxyuridine, is considered 100% identical to AUG, since both are completely complementary to the same sequence (5' -CAU). Examples of alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. One skilled in the art will understand which algorithm to select and which parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally the same length and an expected identity of >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with the standard settings of the Needleman-Wunsch algorithm interface provided by EBI on the web server www.ebi.ac.uk is generally suitable.

- 9 047139 mRNA.

In some embodiments, the composition or composition disclosed herein comprises an mRNA containing an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease or a class 2 Cas nuclease, as described herein. In some embodiments, an mRNA is provided, used, or administered comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease or a class 2 Casnuclease. In some embodiments, the ORF encoding an RNA-guided DNA binding agent is a modified ORF RNA-guided DNA binding agent or simply modified ORF, which are used to briefly indicate that the ORF is modified in one or more of the following ways: (1) the modified ORF has a uridine content ranging from a minimum uridine content to 150% of a minimum uridine content; (2) the modified ORF has a uridine dinucleotide content ranging from a minimum uridine dinucleotide content to 150% of a minimum uridine dinucleotide content; (3) the modified ORF has at least 90% identity with any of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26 , 27, 29, 30, 50, 52, 54, 65 or 66; (4) the modified ORF consists of a set of codons of which at least 75% of the codons are the codon(s) with the minimum uridine content for a given amino acid, e.g., the codon(s) with the fewest uridines (typically 0 or 1, except for the codon for phenylalanine, and this codon with the minimum uridine content has 2 uridines); or (5) the modified ORF contains at least one modified uridine. In some embodiments, the modified OPC is modified by at least two, three, or four of the above methods. In some embodiments, the modified ORF contains at least one modified uridine and is modified by at least one, two, three, or all of (1)-(4) described above.

Modified uridine is used herein to refer to a nucleoside other than thymidine with the same hydrogen bond acceptors as uridine and one or more structural differences from uridine. In some embodiments, the modified uridine is a substituted uridine, that is, a uridine in which one or more non-protic substituents (eg, an alkoxy, such as methoxy) takes the place of a proton. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is a substituted pseudouridine, that is, a pseudouridine in which one or more non-protic substituents (eg, alkyl, such as methyl) takes the place of a proton. In some embodiments, the modified uridine is any of a substituted uridine, a pseudouridine, or a substituted pseudouridine.

As used herein, a uridine position refers to a position in a polynucleotide occupied by uridine or modified uridine. Thus, for example, a polynucleotide in which 100% of the uridine positions are modified uridines contains a modified uridine at every position that would be a uridine in normal RNA (where all bases are standard A, U, C, or G bases) of the same sequence. Unless otherwise indicated, U in a polynucleotide sequence from a sequence table or sequence listing included in or accompanying this specification may be uridine or modified uridine.

Table 1

Codons with minimal uridine content

Amino acid

Codon with minimum content

- 10 047139

uridine A Alanin GCA or GCC or GCG G Glycine GGA or GGC or GGG V Valin GUC or GUA or GUG D Aspartic acid GAC E Glutamic acid GAA or GAG I Isoleucine AUC or AUA or AUG T Threonine ASA or ACC or ACG N Asparagine AAS TO Lysine AAG or AAA S Serin A.G.C. R Arginine AGA or AGG L Leucine CUG or CUA or CUC R Proline CCG or CCA or CCC N G istidine CAC or CAA or CAG Q Glutamine CAG or CAA F Phenylalanine IIS Y Tyrosine UAC WITH Cysteine UGC W Tryptophan UGG m Methionine AUG

In any of the above embodiments, the modified ORF may consist of a set of codons of which at least 75, 80, 85, 90, 95, 98, 99, or 100% of the codons are codons listed in the table of minimum uridine content codons. In any of the above embodiments, the modified ORF may contain a sequence with at least 90, 95, 98, 99 or 100% identity to any of SEQ ID NO: 1,4, 7, 9, 10, 11, 12, 14, 15 , 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65 or 66.

In any of the above embodiments, the modified ORF may contain a sequence with at least 90, 95, 98, 99, or 100% identity to any of SEQ NO: 1, 4, 7, 9, 10, 11, 12, 14, 15 , 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65 or 66.

In any of the above embodiments, the modified ORF may have a uridine content ranging from its minimum uridine content to 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 104, 103, 102, or 101% of the minimum. uridine content.

In any of the above embodiments, the modified ORF may have a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 104, 103, 102, or 101% from the minimum content of uridine dinucleotide.

In any of the above embodiments, the modified ORF may comprise modified uridine at at least one, multiple, or all uridine positions. In some embodiments, the modified uridine is uridine modified at position 5, for example, with a halogen, methyl, or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at position 1, for example, with a halogen, methyl, or ethyl. The modified uridine may be, for example, pseudouridine, M-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is M-methyl-pseudo-uridine. In some embodiments, the modified uridine is a combination of pseudouridine and M-methylpseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1methylpseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and M-methylpseudouridine. In some variants

- 11 047139 in this embodiment, the modified uridine is a combination of pseudouridine and 5iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

In some embodiments, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, or 100% uridine positions in mRNA, as described, are modified uridines. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 5565%, 65-75%, 75-85%, 85-95%, or 90-100 % of the uridine positions in the mRNA are described as modified uridines, for example, 5-methoxyuridine, 5-iodouridine, N1methylpseudouridine, pseudouridine, or a combination thereof. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90 -100% of the uridine positions in the mRNA are reported to be 5-methoxyuridine. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90 -100% of uridine positions in mRNA are reported to be pseudouridine. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90 -100% of the uridine positions in the mRNA are reported to be ω-methylpseudouridine. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90 -100% of the uridine positions in the mRNA are reported to be 5-iodouridine. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90 -100% of the uridine positions in the mRNA are described to be 5-methoxyuridine, and the remainder are 1-methylpseudo-uridine. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90 -100% of the uridine positions in the mRNA are reported to be 5-iodouridine, and the remainder are 1-methylpseudouridine.

In any of the above embodiments, the modified ORF may have a reduced uridine dinucleotide content, such as a minimum possible uridine dinucleotide (UU) content, such as an ORF that (a) uses a codon with a minimum uridine content (as discussed above) at each position and ( b) encodes the same amino acid sequence as the given ORF. Uridine dinucleotide (UU) content can be expressed in absolute units as the sum of UU dinucleotides in the ORF or on a frequency basis as the percentage of positions occupied by uridines in uridine dinucleotides (for example, AUUAU would have a uridine dinucleotide content of 40% because 2 of 5 positions are occupied by uridines of uridine dinucleotide). Modified uridine residues are considered equivalent to uridines for the purpose of estimating minimum uridine dinucleotide content.

In some embodiments, the mRNA comprises at least one untranslated sequence (UTS) from a mammalian expressed mRNA, such as a constitutively expressed mRNA. An mRNA is considered to be constitutively expressed in a mammal if it is continuously transcribed in at least one tissue of a healthy adult mammal. In some embodiments, the mRNA comprises a 5' UTR, a 3' UTR, or a 5' and 3' UTR from a mammalian expressed RNA, such as a constitutively expressed mammalian mRNA. Actin mRNA is an example of a constitutively expressed mRNA.

In some embodiments, the mRNA contains at least one UTR from 17hydroxysteroid beta dehydrogenase 4 (HSD17B4 or HSD), such as the 5' UTR from HSD. In some embodiments, the mRNA comprises at least one UTR of a globin mRNA, such as human alpha-globin (HBA) mRNA, human beta-globin (HBG) mRNA, or Xenopus laevis beta-globin (XBG) mRNA. In some embodiments, the mRNA comprises a 5' UTR, a 3' UTR, or a 5' and 3' UTR from a globin mRNA such as HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 5' UTR from bovine growth hormone, cytomegalovirus (CMV), murine Hba-a1, HSD, albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 3' UTR from bovine growth hormone, cytomegalovirus, murine Hba-a1, HSD, albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises the 5' and 3' UTRs of bovine growth hormone, cytomegalovirus, murine Hba-a1, HSD, albumin gene, HBA, HBB, XBG, heat shock protein 90 (Hsp90), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin, alpha tubulin, tumor protein (p53) or epidermal growth factor receptor (EGFR).

In some embodiments, the mRNA contains 5' and 3' UTRs that are derived from the same source, for example, constitutively expressed mRNA, such as actin, albumin, or globin, such as HBA, HBB, or XBG.

In some embodiments, the mRNA does not contain a 5' UTR, for example, there are no additional nucleotides between the 5' cap and the start codon. In some embodiments, the mRNA contains a Kozak sequence (described below) between the 5' cap and the start codon, but does not have any additional 5' UTR. In some embodiments, the mRNA does not contain a 3' UTR, such as no additional nucleotides between the stop codon and the poly(A) tail.

In some embodiments, the mRNA comprises a Kozak sequence. Follower

- 12 047139 Kozak ability can influence the initiation of translation and the overall yield of the polypeptide translated from mRNA. The Kozak sequence contains a methionine codon that can function as a start codon. The minimal Kozak sequence is NNNRUGN, where at least one of the following is true: the first N is A or G, and the second N is G. In the context of a nucleotide sequence, R is a purine (A or G). In some embodiments, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG. In some embodiments, the Kozak sequence is rccRUGg with zero mismatches or a maximum of one or two mismatches to lowercase positions. In some embodiments, the Kozak sequence is rccAUGg with zero mismatches or a maximum of one or two mismatches to lowercase positions. In some embodiments, the Kozak sequence is gccRccAUGG with zero mismatches or a maximum of one, two, or three mismatches with lowercase positions. In some embodiments, the Kozak sequence is gccAccAUG with zero mismatches or a maximum of one, two, three, or four mismatches with lowercase positions. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG with zero mismatches or a maximum of one, two, three, or four mismatches with lowercase positions.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 43, optionally ORF SEQ ID NO: 43 (i.e., SEQ ID NO: 4) replaced by an alternative ORF by any of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54 , 65 or 66.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 44, optionally ORF SEQ ID NO: 44 (i.e., SEQ ID NO: 4) replaced by an alternative ORF by any of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54 , 65 or 66.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 56, optionally ORF SEQ ID NO: 56 (i.e., SEQ ID NO: 4) replaced by an alternative ORF by any of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54 , 65 or 66.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 57, optionally ORF SEQ ID NO: 57 (i.e., SEQ ID NO: 4) replaced by an alternative ORF by any of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54 , 65 or 66.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: optionally ORF SEQ ID NO: 58 (i.e., SEQ ID NO: 4) replaced by an alternative ORF by any of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65 or 66.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 59, optionally ORF SEQ ID NO: 59 (i.e., SEQ ID NO: 4) replaced by an alternative ORF by any of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54 , 65 or 66.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 60, optionally ORF SEQ ID NO: 60 (i.e., SEQ ID NO: 4) replaced by an alternative ORF by any of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54 , 65 or 66.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 61, optionally ORF SEQ ID NO: 61 (i.e., SEQ ID NO: 4) replaced by an alternative ORF by any of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54 , 65 or 66.

In some embodiments, the mRNA comprises an alternative ORF of any of SEQ ID NO: 7,9, 10, 11, 12, 14, 15, 17, 18,20,21,23,24,26,27,29,30,50, 52, 54, 65 or 66.

In some embodiments, the degree of identity with optionally substituted

- 13 047139 sequences SEQ ID NO: 43, 44 or 56-61 is 95%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NO: 43, 44, or 56-61 is 98%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NO: 43, 44, or 56-61 is 99%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NO: 43, 44, or 56-61 is 100%.

In some embodiments, the mRNA described herein contains a 5' cap, such as cap 0, cap 1, or cap 2. Typically, the 5' cap is a 7-methylguanine ribonucleotide (which may be further modified as discussed below, for example, in relation to ARCA), linked through a 5'-triphosphate to the 5' position of the first nucleotide of the 5'-3' chain of mRNA, i.e. the first cap-proximal nucleotide. In cap 0, the riboses of the first and second cap-proximal nucleotides of the mRNA contain a 2'-hydroxyl. In cap 1, the riboses of the first and second transcribed nucleotides of the mRNA contain 2'-methoxy and 2'-hydroxyl, respectively. In cap 2, the riboses of the first and second capproximal nucleotides of the mRNA contain 2'-methoxy. See, for example, Katibah et al. (2014) Proc Natl Acad Sci USA 111(33): 12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11): E2106–E2115. Most endogenous mRNAs from higher eukaryotes, including mammalian mRNAs such as human mRNA, contain cap 1 or cap 2. Cap 0 and other cap structures other than cap 1 and cap 2 may be immunogenic in mammals such as humans because for recognition as non-self by components of the innate immune system, such as IFIT-1 and IFIT-5, which can lead to increased levels of cytokines, including type I interferon. Components of the innate immune system, such as IFIT-1 and IFIT-5, may also compete with eIF4E to bind mRNA to a cap other than cap 1 or cap 2, potentially inhibiting mRNA translation.

Cap may be included during transcription. For example, ARCA (Orientation Correctly Cap Analog; Thermo Fisher Scientific, cat. no. AM8045) is a cap analog containing 7'-methylguanine 3'-methoxy-5'-triphosphate linked to the 5' position of a guanine ribonucleotide, which may be incorporated in vitro into the transcript during initiation. ARCA results in a cap 0 cap structure in which the 2' position of the first cap-proximal nucleotide is a hydroxyl. See, for example, Stepinski et al., (2001) Synthesis and properties of mRNAs containing the novel 'anti-reverse' cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG , RNA7: 1486-1495. The structure of ARCA is shown below.

_{H Ν} ··\ ' । O P (Ao P-O- G g'YUN < OOO ..... J oUosn, he he

CleanCap™ AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCap™ GG (m7G(5')ppp(5')(2'OMeG )pG; TriLink Biotechnologies, cat. no. N-7133) can be used to provide cap 1 structure during transcription. 3'-O-methylated versions of CleanCap™ AG and CleanCap™ GG are also available from TriLink Biotechnologies as cat. Nos. N-7413 and N-7433, respectively. The structure of CleanCap™ AG is shown below.

Alternatively, the cap may be added to the RNA after transcription. For example, the capping enzyme of vaccinia virus is commercially available (New England Biolabs, cat. no. M2080S) and has RNA triphosphatase and guanylyltransferase activities provided by its D1 subunit, and guanine methyltransferase provided by its D12 subunit. Therefore, it can add 7methylguanine to the RNA, so that cap 0 is formed, in the presence of S-adenosylmethionine and GTP. See, for example, Guo, P. and Moss, W. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994) J. Biol.

- 14 047139

Chem. 269, 24472-24479.

In some embodiments, the mRNA further comprises a polyadenyl (poly(A)) tail. In some embodiments, the poly(A) tail contains at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the poly(A) tail contains 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some cases, the poly(A) tail is interrupted by one or more non-adenine nucleotide anchors at one or more locations within the poly(A) tail. Poly(A) tails may contain at least 8 consecutive adenine nucleotides, but may also contain one or more non-adenine nucleotides. As used herein, the term non-adenine nucleotides refers to any naturally occurring or non-naturally occurring nucleotides that do not contain adenine. Guanine, thymine and cytosine nucleotides are examples of non-adenine nucleotides. Thus, poly(A) tails in the mRNA described herein may contain consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA binding agent or sequence of interest. In some cases, poly-Tails on mRNA contain non-consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA binding agent or sequence of interest, with non-adenine nucleotides interrupting adenine nucleotides at regular or irregularly spaced intervals.

In some embodiments, the mRNA further comprises a polyadenyl (poly(A)) tail. In some embodiments, the poly(A) tail contains at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the poly(A) tail contains 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some cases, the poly(A) tail is interrupted by one or more non-adenine nucleotide anchors at one or more locations within the poly(A) tail. Poly(A) tails may contain at least 8 consecutive adenine nucleotides, but may also contain one or more non-adenine nucleotides. As used herein, the term non-adenine nucleotides refers to any naturally occurring or non-naturally occurring nucleotides that do not contain adenine. Guanine, thymine and cytosine nucleotides are examples of non-adenine nucleotides. Thus, poly(A) tails in the mRNA described herein may contain consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA binding agent or sequence of interest. In some cases, poly-Tails on mRNA contain non-consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA binding agent or sequence of interest, with non-adenine nucleotides interrupting adenine nucleotides at regular or irregularly spaced intervals.

In some embodiments, one or more non-adenine nucleotides are positioned to interrupt consecutive adenine nucleotides such that the poly(A) binding protein can bind to a number of consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides are located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides are located after at least 8-50 consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides are located after at least 8-100 consecutive adenine nucleotides. In some embodiments, the non-adenine nucleotide is located after one, two, three, four, five, six, or seven adenine nucleotides and is followed by at least 8 consecutive adenine nucleotides.

The poly(A) tail may comprise one sequence of consecutive adenine nucleotides followed by one or more non-adenine nucleotides, optionally followed by additional adenine nucleotides.

In some embodiments, the poly(A) tail has or contains one non-adenine nucleotide or one consecutive insertion of 2-10 non-adenine nucleotides. In some embodiments, the non-adenine nucleotide(s) are located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some cases, one or more non-adenine nucleotides are located after at least 8-50 consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides are located after at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 consecutive adenine nucleotides.

In some embodiments, the non-adenine nucleotide is guanine, cytosine, or thymine. In some cases, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is a thymine nucleotide. In some cases where more than one non-adenine nucleotide is present, it is not

- 15 047139 adenine nucleotide can be selected from: a) guanine and thymine nucleotides; b) guanine and cytosine nucleotides; c) thymine and cytosine nucleotides; or d) guanine, thymine and cytosine nucleotides. An exemplary poly(A) tail containing non-adenine nucleotides is shown as SEQ ID NO: 62.

In some embodiments, the mRNA is purified. In some embodiments, the mRNA is purified using a precipitation method (eg, LiCl precipitation, alcohol precipitation, or an equivalent method, for example, as described herein). In some embodiments, the mRNA is purified using a chromatography-based method, such as an HPLC-based method, or an equivalent method (eg, as described herein). In some embodiments, the mRNA is purified using both a precipitation method (eg, LiCl precipitation) and an HPLC-based method.

In some embodiments, at least one gRNA is provided in combination with an mRNA described herein. In some embodiments, the gRNA is provided as a molecule separate from the mRNA. In some embodiments, the gRNA is provided as part, such as part of the UTR of an mRNA described herein.

Chemically modified gRNA.

In some embodiments, the gRNA is chemically modified. A gRNA containing one or more modified nucleosides or nucleotides is called a modified gRNA or chemically modified gRNA to describe the presence of one or more non-naturally occurring and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G residues , C and U. In some embodiments, the modified gRNA is synthesized with a non-canonical nucleoside or nucleotide, referred to herein as modified. Modified nucleosides and nucleotides may include one or more of the following: (i) a change, such as replacing one or both of the non-binding phosphate oxygens and/or one or more bonding phosphate oxygens in the backbone phosphodiester linkage (illustrative backbone modification); (ii) a change, such as replacing a ribose sugar component, such as a 2'-hydroxyl on a ribose sugar (exemplary sugar modification); (iii) complete replacement of the phosphate moiety with dephospho linkers (illustrative backbone modification); (iv) modification or replacement of a naturally occurring nucleic acid base, including a non-canonical nucleic acid base (illustrative base modification); (v) replacing or modifying the ribose phosphate backbone (exemplary backbone modification); (vi) modification of the 3' end or 5' end of the oligonucleotide, such as removal, modification or replacement of a terminal phosphate group or conjugation of a fragment, cap or linker (such modifications of the 3' or 5' cap may include modification of a sugar and/or or skeleton); and (vii) modifying or replacing sugar (illustrative sugar modification).

In some embodiments, the gRNA contains a modified uridine at some or all of the uridine positions. In some embodiments, the modified uridine is uridine modified at position 5, for example, with a halogen or a C1-C6 alkoxy. In some embodiments, the modified uridine is a pseudouridine modified at position 1, for example, with a C1-C6 alkyl. The modified uridine may be, for example, pseudouridine, N-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is 1-methyl-pseudo-uridine. In some embodiments, the modified uridine is a combination of pseudouridine and N-methyl pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5methoxyuridine. In some embodiments, the modified uridine is a combination of N-methylpseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 1-methylpseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

In some embodiments, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, or 100% uridine positions in gRNAs are described as modified uridines. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 5565%, 65-75%, 75-85%, 85-95%, or 90-100 % of the uridine positions in the gRNA are described as modified uridines, for example, 5-methoxyuridine, 5-iodouridine, N1methylpseudouridine, pseudouridine, or a combination thereof. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90 -100% of uridine positions in gRNA are reported to be 5-methoxyuridine. In some embodiments

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10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95% or 90-100% provisions uridines in gRNA are described as pseudouridines. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90 -100% of the uridine positions in the gRNA are described to be ω-methylpseudouridine. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90 -100% of uridine positions in gRNA are reported to be 5-iodouridine. In some embodiments, 10-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 8595%, or 90-100 % of the uridine positions in the gRNA are described to be 5-methoxyuridine and the remainder to be 10-methylpseudouridine. In some embodiments, 1025%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100 % of the uridine positions in the gRNA are described to be 5-iodouridine and the remainder to be N1methylpseudouridine.

Chemical modifications such as those listed above can be combined to produce modified gRNAs containing nucleosides and nucleotides (collectively referred to as residues), which can have two, three, four or more modifications. For example, the modified residue may have a modified sugar and a modified nucleic acid base. In some embodiments, each base of the gRNA is modified, for example, all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all or substantially all of the phosphate groups of the gRNA molecule are replaced with phosphorothioate groups. In some embodiments, the modified gRNAs contain at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified gRNAs contain at least one modified residue at or near the 3' end of the RNA.

In some embodiments, the gRNA contains one, two, three, or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions in the modified gRNA are modified nucleosides or nucleotides.

Unmodified nucleic acids may be subject to degradation, for example by intracellular nucleases or nucleases found in serum. For example, nucleases can hydrolyze phosphodiester bonds of nucleic acids. Accordingly, in one aspect, the gRNAs described herein may contain one or more modified nucleosides or nucleotides, for example, to impart stability to intracellular or serum nucleases. In some embodiments, modified gRNA molecules described herein can cause a reduced innate immune response when introduced into a population of cells both in vivo and ex vivo. The term innate immune response includes the cellular response to exogenous nucleic acids, including single-stranded nucleic acids, which includes the induction of expression and release of cytokines, especially interferons, and cell death.

In some backbone modification embodiments, the phosphate group of the modified moiety may be modified by replacing one or more oxygen atoms with another substituent. In addition, a modified residue, such as a modified residue present in a modified nucleic acid, may involve complete replacement of an unmodified phosphate group with a modified phosphate group, as described herein. In some embodiments, modification of the phosphate backbone may include changes that result in either an uncharged linker or a charged linker with an asymmetric charge distribution.

Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrophosphonates, phosphoramidates, alkyl or arylphosphonates, and phosphotriesters. The phosphorus atom in the unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygen atoms with one of the above atoms or groups of atoms can make the phosphorus atom chiral. A stereogenic phosphorus atom may have either an R configuration (herein Rp) or an S configuration (herein Sp). The backbone can also be modified by replacing the bridging oxygen (that is, the oxygen that links the phosphate to the nucleoside) with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene phosphonates). The replacement can occur at either the linker oxygen or both linker oxygens.

In some modifications of the backbone, the phosphate group can be replaced by connecting groups that do not contain phosphorus. In some embodiments, the charged phosphate group may be replaced by a neutral moiety. Examples of moieties that may replace the phosphate group may include, but are not limited to, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formoacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

Template nucleic acid.

The compositions and methods described herein may include a template nucleic acid. The template can be used to modify or insert a nucleic acid sequence at or near a Cas nuclease target site. In some embodiments, the methods include introducing a matrix into a cell. In some embodiments, a single matrix may be provided. In other embodiments, two or more matrices may be provided so that editing can occur at two or more target sites. For example, different templates may be proposed for editing one gene in a cell or two different genes in a cell.

In some embodiments, the template may be used in homologous recombination. In some embodiments, homologous recombination may result in integration of a template sequence or portion of a template sequence into a target nucleic acid molecule. In other embodiments, the template may be used in homology-directed repair, which involves introducing a DNA strand at a cleavage site in a nucleic acid. In some embodiments, homology-directed repair may result in the inclusion of a template sequence in the edited target nucleic acid molecule. In yet other embodiments, the template can be used in non-homologous end joining-mediated gene editing. In some embodiments, the template sequence has no similarity to a nucleic acid sequence near the cleavage site. In some embodiments, a matrix or a portion of a matrix sequence is included. In some embodiments, the template comprises flanking inverted terminal repeat (ITR) sequences.

In some embodiments, the template may comprise a first homology arm and a second homology arm (also referred to as a first and second nucleotide sequence) that are complementary to sequences located upstream and downstream of the cleavage site, respectively. When the matrix contains two homology arms, each arm may be the same length or different lengths, and the sequence between the homology arms may be substantially similar or identical to the target sequence between the homology arms, or it may be completely unrelated. In some embodiments, the degree of complementarity or percentage identity between the first nucleotide sequence on the template and the sequence upstream of the cleavage site and between the second nucleotide sequence on the template and the sequence downstream of the cleavage site may allow for homologous recombination, such as, for example, homologous recombination high precision between the template and the target nucleic acid molecule. In some embodiments, the degree of complementarity may be about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100%. In some embodiments, the degree of complementarity may be about 95, 97, 98, 99, or 100%. In some embodiments, the degree of complementarity may be at least 98, 99, or 100%. In some embodiments, the degree of complementarity may be 100%. In some embodiments, the percent identity may be about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100%. In some embodiments, the percent identity may be about 95, 97, 98, 99, or 100%. In some embodiments, the percent identity may be at least 98%, 99%, or 100%. In some embodiments, the percent identity may be 100%.

In some embodiments, the template sequence may match, contain, or consist of an endogenous sequence of the target cell. It may also or alternatively correspond to, contain or consist of an exogenous sequence of the target cell. As used herein, the term endogenous sequence refers to a sequence that is native to a cell. The term exogenous sequence refers to a sequence that is not native to a cell, or to a sequence whose native location in the cell's genome is elsewhere. In some embodiments, the endogenous sequence may be the genomic sequence of a cell. In some embodiments, the endogenous sequence may be a chromosomal or extrachromosomal sequence. In some embodiments, the endogenous sequence may be a plasmid sequence of the cell. In some embodiments, the template sequence may be substantially identical to a portion of the endogenous sequence in the cell at or near the cleavage site, but contain at least one nucleotide change. In some embodiments, editing a cleaved target nucleic acid molecule using a template may result in an insertion mutation,

- 18 047139 deletion or substitution of one or more nucleotides of the target nucleic acid molecule. In some embodiments, the mutation may result in one or more amino acid changes in the protein expressed by the gene containing the target sequence. In some embodiments, the mutation may result in one or more nucleotide changes in the RNA expressed by the target gene. In some embodiments, the mutation may change the level of expression of the target gene. In some embodiments, the mutation may result in increased or decreased expression of the target gene. In some embodiments, the mutation may result in gene knockdown. In some embodiments, the mutation may result in a gene knockout. In some embodiments, the mutation may result in restoration of gene function. In some embodiments, editing a cleaved target nucleic acid molecule by a template may result in a change in the exon sequence, intron sequence, regulatory sequence, transcriptional control sequence, translational control sequence, splice site, or non-coding sequence of the target nucleic acid molecule, such as DNA.

In other embodiments, the template sequence may comprise an exogenous sequence. In some embodiments, the exogenous sequence may comprise a protein or RNA coding sequence operably linked to the exogenous promoter sequence such that upon integration of the exogenous sequence into a target nucleic acid molecule, the cell is capable of expressing the protein or RNA encoded by the integrated sequence. In other embodiments, after integration of an exogenous sequence into a target nucleic acid molecule, expression of the integrated sequence may be regulated by an endogenous promoter sequence. In some embodiments, the exogenous sequence may provide a cDNA sequence encoding a protein or portion of a protein. In still other embodiments, the exogenous sequence may comprise or consist of an exon sequence, an intron sequence, a regulatory sequence, a transcriptional control sequence, a translational control sequence, a splice site, or a non-coding sequence. In some embodiments, integration of an exogenous sequence may result in restoration of gene function. In some embodiments, integration of an exogenous sequence may result in gene knockin. In some embodiments, integration of an exogenous sequence may result in gene knockout.

The matrix can be of any suitable length. In some embodiments, the matrix may contain 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000 or more nucleotides in length. The template may be a single-stranded nucleic acid. The template may be a double-stranded or partially double-stranded nucleic acid. In certain embodiments, the length of the single-stranded template is 20, 30, 40, 50, 75, 100, 125, 150, 175, or 200 nucleotides. In some embodiments, the template may contain a nucleotide sequence that is complementary to a portion of the target nucleic acid molecule containing the target sequence (ie, a homology arm). In some embodiments, the template may contain a homology arm that is complementary to a sequence located upstream and downstream of a cleavage site on the target nucleic acid molecule.

In some embodiments, the template comprises ssDNA or dsDNA containing inverted terminal repeat (ITR) flanking sequences. In some embodiments, the template is provided as a vector, plasmid, minicircle, nanocircuit, or PCR product.

Purification of nucleic acids.

In some embodiments, the nucleic acid is purified. In some embodiments, the nucleic acid is purified using a precipitation method (eg, LiCl precipitation, alcohol precipitation, or an equivalent method, for example, as described herein). In some embodiments, the nucleic acid is purified using a chromatography-based method, such as an HPLC-based method, or an equivalent method (eg, as described herein). In some embodiments, the nucleic acid is purified using both a precipitation method (eg, LiCl precipitation) and an HPLC-based method.

Target sequences.

In some embodiments, the CRISPR/Cas system as described herein can be directed to and cleave a target sequence in a target nucleic acid molecule. For example, the target sequence can be recognized and cleaved by Cas nuclease. In certain embodiments, the target sequence for a Cas nuclease is located adjacent to a related PAM nuclease sequence. In some embodiments, a class 2 Casnuclease may be directed by a gRNA to a target sequence of a target nucleic acid molecule, where the gRNA hybridizes to the target sequence and the Cas protein

- 19 047139 class 2 splits it. In some embodiments, the guide RNA is hybridized and a class 2 Casnuclease cleaves the target sequence adjacent to or containing its cognate PAM. In some embodiments, the target sequence may be complementary to the guide RNA targeting sequence. In some embodiments, the degree of complementarity between the guide RNA targeting sequence and the portion of the corresponding target sequence that hybridizes to the guide RNA may be about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98. 99 or 100%. In some embodiments, the percentage identity between the guide RNA targeting sequence and the portion of the corresponding target sequence that hybridizes to the guide RNA may be about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98. 99 or 100%. In some embodiments, the region of homology of the target is adjacent to a related PAM sequence. In some embodiments, the targeting sequence may comprise a sequence that is 100% complementary to the guide RNA targeting sequence. In other embodiments, the target sequence may contain at least one mismatch, deletion, or insertion compared to the guide RNA targeting sequence.

The length of the target sequence may depend on the nuclease system used. For example, a guide RNA targeting sequence for a CRISPR/Cas system may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length, and the target sequence has a corresponding length, not necessarily adjacent to the PAM sequence. In some embodiments, the target sequence may be 15-24 nucleotides in length. In some embodiments, the target sequence may be 17-21 nucleotides in length. In some embodiments, the target sequence may be 20 nucleotides in length. When nickases are used, the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave opposite strands of the DNA molecule. In some embodiments, the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave the same strands of a DNA molecule. In some embodiments, the target sequence may comprise a portion of target sequences recognized by one or more Cas nucleases.

The target nucleic acid molecule can be any DNA or RNA molecule that is endogenous or exogenous to the cell. In some embodiments, the target nucleic acid molecule may be episomal DNA, plasmid, genomic DNA, viral genome, mitochondrial DNA, or chromosomal DNA from or in a cell. In some embodiments, the target sequence of the target nucleic acid molecule may be a genomic sequence from or in a cell, including a human cell.

In additional embodiments, the target sequence may be a viral sequence. In other embodiments, the target sequence may be a pathogen sequence. In yet other embodiments, the target sequence may be a synthesized sequence. In additional embodiments, the target sequence may be a chromosomal sequence. In certain embodiments, the target sequence may comprise a translocation insertion, such as a cancer-associated translocation. In some embodiments, the target sequence may be located on a eukaryotic chromosome, such as a human chromosome. In certain embodiments, the target sequence is a liver-specific sequence at which it is expressed in liver cells.

In some embodiments, the target sequence may be located within the coding sequence of a gene, the intronic sequence of a gene, a regulatory sequence, a transcriptional control sequence of a gene, a translational control sequence of a gene, a splice site, or a noncoding sequence between genes. In some embodiments, the gene may be a gene encoding a protein. In other embodiments, the gene may be a gene that does not code for RNA. In some embodiments, the target sequence may comprise all or a portion of a disease-associated gene. In some embodiments, the target sequence may be located at a nongenic functional site in the genome, such as a site that controls aspects of chromatin organization, such as a scaffolding site or locus regulatory regions.

In embodiments involving a Cas nuclease, such as a class 2 nuclease, the target sequence may be adjacent to a protospacer adjacent motif (PAM). In some embodiments, PAM may be adjacent to or within 1, 2, 3, or 4 nucleotides of the 3' end of the sequence. The length and sequence of PAM may depend on the Cas protein used. For example, the PAM may be selected from a consensus or specific PAM sequence for

- 20047139 a specific Cas9 protein or Cas9 orthologue, including those shown in FIG. 1 from Ran et al., Nature, 520: 186-191 (2015) and FIG. S5 from Zetsche 2015, the corresponding description of each of which is incorporated herein by reference. In some embodiments, the PAM may be 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG, NGGNG, NG, NAAAAN, NNAAAAW, NNNNACA, GNNNCNNA, TTN, and NNNNGATT (where N is defined as any nucleotide and W is defined as either A or T). In some embodiments, the PAM sequence may be NGG. In some embodiments, the PAM sequence may be NGGNG. In some embodiments, the PAM sequence may be TTN. In some embodiments, the PAM sequence may be NNAAAAW.

Lipid composition.

This document describes various options for RNA LNP formulations, including CRISPR/Cas cargo. Such LNP formulations contain an amino lipid along with an auxiliary lipid, a neutral lipid and a PEG lipid. In some embodiments, such LNP formulations contain an amino lipid along with an auxiliary lipid and a PEG lipid. In some embodiments, the LNP formulations contain less than 1 percent neutral phospholipid. In some embodiments, the LNP formulations contain less than 0.5 percent neutral phospholipid. By lipid nanoparticle is meant a particle that contains multiple (ie, more than one) lipid molecules physically bound to each other by intermolecular forces.

Aminolipids.

LNP compositions for the delivery of bioactive agents contain an aminolipid, which is defined as Lipid A or its equivalents, including acetal analogues of Lipid A.

In some embodiments, the aminolipid is Lipid A, which is (9Z, 12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3(diethylamino)propoxy)carbonyl) oxy)methyl)propylooctadeca-9,12-dienoate, also called 3((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 122)-octadeca-9,12-dienoate.

Lipid A can be depicted as:

ABOUT

Lipid A can be synthesized according to WO 2015/095340 (eg pages 84-86). In certain embodiments, the aminolipid is the equivalent of Lipid A.

In certain embodiments, the aminolipid is a Lipid A analog. In certain embodiments, the Lipid A analog is an acetal analog of Lipid A. In certain LNP compositions, the acetal analog is a C4-C12 acetal analog. In some embodiments, the acetal analogue is a C5-C12 acetal analogue. In additional embodiments, the acetal analogue is a C5-C10 acetal analogue. In other embodiments, the acetal analogue is selected from C4, C5, C6, C7, C9, C10, C11 and C12 acetal analogue.

Aminolipids suitable for use in the LNPs described herein are biodegradable in vivo and useful for delivering a biologically active agent, such as RNA, into a cell. Aminolipids have low toxicity (eg, tolerated in animal models without harmful effects at levels greater than or equal to 10 mg/kg RNA cargo). In certain embodiments, aminolipid-containing LNPs include those for which at least 75% of the aminolipid is cleared from plasma within 8, 10, 12, 24, or 48 hours or 3, 4, 5, 6, 7, or 10 days. In certain embodiments, aminolipid-containing LNPs include those for which at least 50% of the mRNA or gRNA is cleared from plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. In certain embodiments, aminolipid-containing LNPs include those for which at least 50% of the LNP is cleared from plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. for example, by measuring a lipid (eg, aminolipid), RNA (eg, mRNA), or other component. In certain embodiments, the lipid-encapsulated and lipid-free RNA or nucleic acid component of the LNP is measured.

Lipid clearance can be measured as described in the literature. See Maier, M.A., et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78 (Maier). For example, at Maier, LNP-siRNA systems containing siRNA targeting luciferases were administered to six- to eight-week-old male C57B1/6 mice at a dose of 0.3 mg/kg by intravenous bolus injection through the lateral tail vein. Blood samples,

- 21 047139 livers and spleens were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96 and 168 hours after dosing. Mice were perfused with saline before tissue collection, and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC/MS. In addition, Maier describes a procedure for assessing toxicity following administration of LNP-siRNA formulations. For example, siRNA targeting luciferase was administered at doses of 0, 1, 3, 5 and 10 mg/kg (5 animals per group) via a single intravenous bolus of 5 ml/kg dose to male SpregDawley rats. After 24 hours, approximately 1 ml of blood was obtained from the jugular vein of awake animals and the serum was isolated. 72 hours after dosing, all animals were sacrificed for necropsy. Clinical manifestations, body weight, serum chemistry, organ weights, and histopathology were assessed. Although Maier describes methods for evaluating siRNA-LNP formulations, these methods can be used to evaluate the clearance, pharmacokinetics and toxicity of administration of LNP compositions according to this invention.

Aminolipids may result in increased clearance rates. In some embodiments, the clearance rate is the rate of lipid clearance, such as the rate at which a lipid is cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the rate of RNA clearance, such as the rate at which mRNA or gRNA is cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is cleared from tissue, such as liver tissue or spleen tissue. In certain embodiments, a high clearance rate results in a safety profile without significant side effects. Aminolipids may reduce the accumulation of LNP in the circulatory system and tissues. In some embodiments, reduced accumulation of LNP in the circulatory system and tissues results in a safety profile without significant side effects.

Aminolipids as described herein are ionizable (eg, can form a salt) depending on the pH of the environment in which they are found. For example, in a slightly acidic environment, aminolipids can be protonated and thus carry a positive charge. Conversely, in a weakly basic environment such as blood, where the pH is approximately 7.35, aminolipids may not be protonated and therefore not carry a charge. In some embodiments, the aminolipids herein may be protonated at a pH of at least about 9. In some embodiments, the aminolipids herein may be protonated at a pH of at least about 9. In some embodiments, the aminolipids herein may be protonated at pH at least about 10.

The pH at which an aminolipid is predominantly protonated is related to its own pKa. In some embodiments, the aminolipids of this invention may each independently have a pKa ranging from about 5.1 to about 7.4. In some embodiments, the aminolipids of this invention may each independently have a pKa in the range of about 5.5 to about 6.6. In some embodiments, the aminolipids of this invention may each independently have a pKa in the range of about 5.6 to about 6.4. In some embodiments, the aminolipids of this invention may each independently have a pKa in the range of about 5.8 to about 6.2. For example, the aminolipids of this invention may each independently have a pKa in the range of about 5.8 to about 6.5. The pKa value of the amino lipid may be an important factor in LNP formulation, as cationic lipids with a pKa in the range of about 5.1 to about 7.4 have been found to be effective for delivering cargo in vivo, such as to the liver. In addition, cationic lipids with a pKa in the range of about 5.3 to about 6.4 have been found to be effective for delivery in vivo, such as to tumors. See for example WO 2014/136086.

Additional lipids.

Neutral lipids suitable for use in the lipid composition as described herein include, for example, various neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use herein include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC) , phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (ePC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2- palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2diarachigoyl-sn-glycero-3-phosphocholine (DBPC) 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC) . dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be

- 22 047139 is selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC). In another embodiment, the neutral phospholipid may be dipalmitoylphosphatidylcholine (DPPC).

Accessory lipids include sterols, sterols and alkylresorcinols.

Auxiliary lipids suitable for use herein include, but are not limited to, cholesterol, 5-heptadecylresorcinol and cholesterol hemisuccinate. In one embodiment, the auxiliary lipid may be cholesterol. In one embodiment, the auxiliary lipid may be cholesterol hemisuccinate.

PEG lipids are stealth lipids that alter the length of time that nanoparticles can survive in vivo (e.g., in the blood). PEG lipids can assist in the formulation process, for example by reducing particle aggregation and controlling particle size. The PEG lipids used herein can modulate the pharmacokinetic properties of LNP. Typically, a PEG lipid contains a lipid moiety and a PEG-based polymer moiety.

In some embodiments, the lipid moiety may be derived from a diacylglycerol or diacylglycamide, including those containing a dialkylglycerol or dialkylglycamide group having an alkyl chain length independently containing from about C4 to about C40 saturated or unsaturated carbon atoms, which chain may contain one or more functional groups such as, for example, amide or ester. In some embodiments, the alkyl chain length is from about C10 to C20. The dialkylglycerol or dialkylglycamide group may further contain one or more substituted alkyl groups. The length of the chain can be symmetrical or asymmetrical.

Unless otherwise specified, the term PEG as used herein means any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, the PEG moiety is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety is alternatively the PEG moiety may be substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment, the PEG moiety includes a PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, for example, J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); alternatively, the PEG moiety does not include PEG copolymers, for example it may be a PEG monopolymer. In one embodiment, the PEG has a molecular weight of about 130 to about 50,000, in a sub-embodiment from about 150 to about 30,000, in a sub-embodiment from about 150 to about 20,000, in a sub-embodiment from about 150 to about 15,000, in a sub-embodiment from about 150 to about 10,000, in a sub-embodiment from about 150 to about 6,000, in a sub-embodiment from about 150 to about 5,000, in a sub-embodiment from about 150 to about 4,000 in a sub-embodiment from about 150 to about 3,000, in a sub-embodiment from about 300 to about 3000, in a sub-embodiment from about 1000 to about 3000, and in a sub-embodiment from about 1500 to about 2500.

In certain embodiments, the PEG (eg, conjugated to a lipid moiety or lipid, such as a stealth lipid) is PEG-2K, also called PEG 2000, which has an average molecular weight of about 2000 daltons. PEG-2K is represented herein by the following formula (I), where n is 45, which means that the number average degree of polymerization (I) is about 45 ⁿ subunits. However, other PEG embodiments known in the art may be used, including, for example, those where the number average degree of polymerization is about 23 subunits (n=23) and/or 68 subunits (n=68). In some embodiments, n can range from about 30 to about 60. In some embodiments, n can range from about 35 to about 55. In some embodiments, n can range from about 40 to about 50. In some embodiments, n can range from about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.

In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (Cat. No. GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol . -5-ene-3 [beta]oxy)carboxamido-3', 6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4

- 23 047139 ditetradecoxybenzyl-[omega]-methylpoly(ethylene glycol) ether), 1,2-dimyristoyl^p-glycero-3phosphoethanolamine^-[methoxy(polyethylene glycol)-2000| (PEG2k-DMG) (Cat. No. 880150P from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-glycero-3-phosphoethanolamine-N [methoxy(polyethylene glycol)-2000] (PEG2k -DSPE) (Cat. No. 880120C from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-p-glycerol. methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF, Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA) and 1,2 distearyloxypropyl-.3-amine^-[methoxy(polyethylene glycol)-2000 | (PEG2c-DSA). In one embodiment, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In one embodiment of the present invention, the PEG lipid may be PEG2k-DSPE. In one embodiment, the PEG lipid may be PEG2k-DMA. In one embodiment, the PEG lipid may be PEG2k-C-DSPE. In one embodiment, the PEG lipid may be compound S027, described in WO2016/010840 in paragraphs [00240] to [00244]. In one embodiment, the PEG lipid may be PEG2k-DSA. In one embodiment, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18.

LNP formulations.

Embodiments provide lipid compositions described in accordance with the respective molar ratios of the lipid components in the composition. In one embodiment, the mol.% of the aminolipid may be from about 30 to about 60 mol.% mol.%. In one embodiment, the mol % aminolipid may be from about 40 to about 60 mol %. In one embodiment, the mol % aminolipid may be from about 45 to about 60 mol %. In one embodiment, the mol % aminolipid may be from about 50 to about 60 mol %. In one embodiment, the mol % of the aminolipid may be from about 55 to about 60 mol %. In one embodiment, the mol % aminolipid may be from about 50 to about 55 mol %. In one embodiment, the mol % aminolipid may be about 50 mol %. In one embodiment, the mol % aminolipid may be about 55 mol %. In some embodiments, the aminolipid mol% in the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol%. In some embodiments, the mol% aminolipid in the LNP batch will be ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol .% mol.% or ±0.25 mol.% of the target mol.%. All mol% numbers are given as the proportion of lipid component in the LNP compositions. In certain embodiments, the batch-to-batch variability of the amino lipid mol% LNP will be less than 15%, less than 10%, or less than 5%.

In one embodiment, the mole percent of the neutral lipid, such as a neutral phospholipid, may be from about 5 to about 15 mole percent. In one embodiment, the mole % of the neutral lipid, such as a neutral phospholipid, may be from about 7 to about 12 mole %. In one embodiment, the mole % of the neutral lipid, such as a neutral phospholipid, may be from about 0 to about 5 mole % . In one embodiment, the mol % of the neutral lipid, such as a neutral phospholipid, may be from about 0 mol % to about 10 mol %. In one embodiment, the mol % of the neutral lipid, such as a neutral phospholipid, may be from about 5 to about 10 mol.%. In one embodiment, the mole% of the neutral lipid, such as a neutral phospholipid, can be from about 8 mole% to about 10 mole%.

In one embodiment, the mol % of the neutral lipid, such as a neutral phospholipid, may be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 mol. %. In one embodiment, the mole% neutral lipid, such as a neutral phospholipid, may be about 9 mole%.

In one embodiment, the mole percent of the neutral lipid, such as a neutral phospholipid, may be from about 1 to about 5 mole percent. In one embodiment, the mol % of the neutral lipid may be from about 0.1 to about 1 mol %. In one embodiment, the mole % of the neutral lipid, such as a neutral phospholipid, may be about 0.1, about 0.2, about 0.5, 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5 or about 5 mol%.

In one embodiment, the mole percent of the neutral lipid, such as a neutral phospholipid, may be less than about 1 mole percent. In one embodiment, the mol% of the neutral lipid, such as a neutral phospholipid, may be less than 0.5 mol%. In one embodiment, the mole% of the neutral lipid, such as a neutral phospholipid, may be about 0 mole%, about 0.1 mole%, about 0.2 mole%, about 0.3 mole%, about 0.4 mol.%, about 0.5 mol.%, about 0.6 mol.%, about 0.7 mol.%, about 0.8 mol.%, about 0.9 mol.%, or about 1 mol.%. In some embodiments, the compositions described herein do not contain neutral lipid (ie, 0 mol.% neutral lipid). In some embodiments, the compositions described in this

- 24 047139 document, essentially do not contain neutral lipid (ie about 0 mol.% neutral lipid). In some embodiments, the compositions described herein do not contain neutral phospholipid (ie, 0 mol% neutral phospholipid). In some embodiments, the compositions described herein contain substantially no neutral phospholipid (ie, about 0 mole percent neutral phospholipid).

In some embodiments, the mole% neutral lipid in the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mole% neutral lipid . In certain embodiments, the LNP lot-to-lot variability will be less than 15%, less than 10%, or less than 5%.

In one embodiment, the mol % of the auxiliary lipid may be from about 20 mol % to about 60 mol %. In one embodiment, the mol % of the auxiliary lipid can be from about 25 mol % to about 55 mol %. In one embodiment, the mol % of the auxiliary lipid may be from about 25 mol % to about 50 mol %. In one embodiment, the mol % of the auxiliary lipid may be from about 25 mol % to about 40 mol %. In one embodiment, the mol % of the auxiliary lipid may be from about 30 mol % to about 50 mol %. In one embodiment, the mol % of the auxiliary lipid may be from about 30 mol % to about 40 mol %. In one embodiment, the mol% of the auxiliary lipid is adjusted based on the aminolipid, neutral lipid, and PEG lipid concentrations to bring the lipid component to 100 mol%. In one embodiment, the mol% of the auxiliary lipid is adjusted based on the aminolipid and PEG lipid concentrations to bring the lipid component to 100 mol%. In one embodiment, the mol% of the auxiliary lipid is adjusted based on the aminolipid and PEG concentrations to bring the lipid component to 99 mol%. In some embodiments, the mol% of the auxiliary lipid in the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol%. In certain embodiments, the LNP lot-to-lot variability will be less than 15%, less than 10%, or less than 5%.

In one embodiment, the mol % of the PEG lipid may be from about 1 to about 10 mol %. In one embodiment, the mol % of the PEG lipid may be from about 2 to about 10 mol %. In one embodiment, the mol % of the PEG lipid may be from about 2 to about 8 mol %. In one embodiment, the mol % of the PEG lipid may be from about 2 to about 4 mol %. In one embodiment, the mol % of the PEG lipid may be from about 2.5 to about 4 mol %. In one embodiment, the mol % of the PEG lipid may be about 3 mol %. In one embodiment, the mol % of the PEG lipid may be about 2.5 mol %. In some embodiments, the mole% of the PEG lipid in the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target PEG mole% -lipid. In certain embodiments, the LNP lot-to-lot variability will be less than 15%, less than 10%, or less than 5%.

In certain embodiments, the cargo comprises an mRNA encoding an RNA-guided DNA binding agent (e.g., a Cas nuclease, class 2 Cas nuclease, or Cas9), and a gRNA or a nucleic acid encoding a gRNA, or a combination of mRNA and gRNA. In one embodiment, the LNP composition may contain Lipid A or equivalents thereof. In some aspects, the aminolipid is Lipid A. In some aspects, the aminolipid is an equivalent of Lipid A, such as a Lipid A analog. In some aspects, the aminolipid is an acetal analog of Lipid A. In various embodiments, the LNP composition comprises an aminolipid, a neutral lipid, an auxiliary lipid, and PEG lipid. In certain embodiments, the auxiliary lipid is cholesterol. In certain embodiments, the neutral lipid is DSPC. In specific embodiments, the PEG lipid is PEG2k-DMG. In some embodiments, the LNP composition may contain Lipid A, an auxiliary lipid, a neutral lipid, and a PEG lipid. In some embodiments, the LNP composition contains an amino lipid, DSPC, cholesterol, and a PEG lipid. In some embodiments, the LNP composition contains a PEG lipid containing DMG. In certain embodiments, the aminolipid is selected from Lipid A and a Lipid A equivalent, including an acetal analogue of Lipid A. In further embodiments, the LNP composition contains Lipid A, cholesterol, DSPC, and PEG2k-DMG.

In various embodiments, the LNP composition contains an amino lipid, an auxiliary lipid, a neutral lipid, and a PEG lipid. In various embodiments, the LNP composition comprises an amino lipid, an auxiliary lipid, a neutral phospholipid, and a PEG lipid. In various embodiments, the LNP composition contains a lipid component that consists of an amino lipid, an auxiliary lipid, a neutral lipid, and a PEG lipid. In various embodiments, the LNP composition contains an amino lipid, an auxiliary lipid, and a PEG lipid. In certain embodiments, the LNP composition does not contain a neutral lipid, such as a neutral phospholipid. In various embodiments, the LNP composition contains a lipid component that consists of an amino lipid, an auxiliary lipid, and a PEG lipid. In certain variants

- 25 047139 implementation of the neutral lipid is selected from one or more of DSPC, DPPC, DAPC, DMPC, DOPC, DOPE, and DSPE. In certain embodiments, the neutral lipid is DSPC. In certain embodiments, the neutral lipid is DPPC. In certain embodiments, the neutral lipid is DAPC. In certain embodiments, the neutral lipid is DMPC. In certain embodiments, the neutral lipid is DOPC. In certain embodiments, the neutral lipid is DOPE. In certain embodiments, the neutral lipid is DSPE. In certain embodiments, the auxiliary lipid is cholesterol. In specific embodiments, the PEG lipid is PEG2k-DMG. In some embodiments, the LNP composition may contain Lipid A, an auxiliary lipid, and a PEG lipid. In some embodiments, the LNP composition may contain a lipid component that consists of Lipid A, an auxiliary lipid, and a PEGlipid. In some embodiments, the LNP composition contains an amino lipid, cholesterol, and a PEG lipid. In some embodiments, the LNP composition contains a lipid component that consists of an amino lipid, cholesterol, and a PEG lipid. In some embodiments, the LNP composition contains a PEG lipid containing DMG. In certain embodiments, the aminolipid is selected from Lipid A and a Lipid A equivalent, including an acetal analogue of Lipid A. In certain embodiments, the aminolipid is a C5-C12 or C4-C12 acetal analogue of Lipid A. In further embodiments, the LNP composition comprises Lipid A, cholesterol and PEG2k-DMG.

Embodiments of the present invention also provide lipid compositions described in accordance with the molar ratio between the positively charged amino groups of the aminolipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This can be represented mathematically by the N/P equation. In some embodiments, the LNP composition may contain a lipid component that contains an amino lipid, an auxiliary lipid, a neutral lipid, and a PEG lipid; and a nucleic acid component, wherein the N/P ratio is from about 3 to 10. In some embodiments, the LNP composition may contain a lipid component that contains an amino lipid, an auxiliary lipid, and a PEG lipid; and a nucleic acid component, wherein the N/P ratio is from about 3 to 10. In some embodiments, the LNP composition may contain a lipid component that contains an amino lipid, an auxiliary lipid, a neutral lipid, and an auxiliary lipid; and an RNA component, wherein the N/P ratio is from about 3 to 10. In some embodiments, the LNP composition may contain a lipid component that contains an amino lipid, an auxiliary lipid, and a PEG lipid; and an RNA component, wherein the N/P ratio is from about 3 to 10. In one embodiment, the N/P ratio can be from about 5 to 7. In one embodiment, the N/P ratio can be from about 3 to 7. In In one embodiment, the N/P ratio may be from about 4.5 to 8. In one embodiment, the N/P ratio may be about 6. In one embodiment, the N/P ratio may be 6 ± 1. In one embodiment, the N/P ratio may be 6 ± 0.5. In some embodiments, the N/P ratio will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target N/P ratio. In certain embodiments, the LNP lot-to-lot variability will be less than 15%, less than 10%, or less than 5%.

In some embodiments, the nucleic acid component, such as the RNA component, may comprise mRNA, such as mRNA encoding a Cas nuclease. The RNA component includes RNA, optionally with additional nucleic acid and/or protein, for example, RNPcargo. In one embodiment, the RNA comprises Cas9 mRNA. In some compositions containing mRNA encoding a Cas nuclease, the LNP further comprises a gRNA nucleic acid, such as gRNA. In some embodiments, the RNA component comprises Cas nuclease mRNA and gRNA. In some embodiments, the RNA component comprises a class 2 Cas nuclease mRNA and a gRNA.

In certain embodiments, the LNP composition may contain mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, an amino lipid, an auxiliary lipid, a neutral lipid, and a PEG lipid. In certain embodiments, the LNP composition may contain mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, an amino lipid, an auxiliary lipid, and a PEG lipid. In some LNP compositions containing mRNA encoding a Cas nuclease, such as class 2 Cas nuclease, the accessory lipid is cholesterol. In other compositions containing mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, the neutral lipid is DSPC. In further embodiments, comprising mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, the PEG lipid is PEG2k-DMG or PEG2k-C11. In particular compositions containing mRNA encoding a Cas nuclease, such as a class 2 Casnuclease, the aminolipid is selected from Lipid A and its equivalents, such as an acetal analog

- 26 047139

Lipida A.

In some embodiments, the LNP composition may comprise gRNA. In certain embodiments, the LNP composition may comprise an amino lipid, a gRNA, an auxiliary lipid, a neutral lipid, and a PEG lipid. In certain embodiments, the LNP composition may comprise an amino lipid, a gRNA, an auxiliary lipid, and a PEG lipid. In some LNP compositions containing gRNA, the accessory lipid is cholesterol. In some compositions containing gRNA, the neutral lipid is DSPC. In additional embodiments containing gRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the aminolipid is selected from Lipid A and its equivalents, such as an acetal analogue of Lipid A.

In one embodiment, the LNP composition may contain ssRNA. In one embodiment, the LNP composition may contain a Cas9 sRNA. In one embodiment, the LNP composition may contain a Cpf1 sRNA. In some compositions containing ssRNA, the LNP contains an amino lipid, an auxiliary lipid, a neutral lipid, and a PEG lipid. In some compositions containing ssRNA, the LNP contains an amino lipid, an auxiliary lipid, and a PEG lipid. In certain compositions containing ssRNA, the accessory lipid is cholesterol. In other compositions containing ssRNA, the neutral lipid is DSPC. In additional embodiments containing ssRNA, the PEG lipid is PEG2k-DMG or PEG2kC11. In certain embodiments, the aminolipid is selected from Lipid A and its equivalents, such as acetal analogues of Lipid A.

In certain embodiments, the LNP composition contains an mRNA encoding a Casnuclease and a gRNA, which may be an sRNA. In one embodiment, the LNP composition may contain an aminolipid, mRNA encoding a Cas nuclease, gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In one embodiment, the LNP composition may contain a lipid component consisting of an amino lipid, an auxiliary lipid, a neutral lipid, and a PEG lipid; and a nucleic acid component consisting of an mRNA encoding a Cas nuclease and a gRNA. In one embodiment, the LNP composition may contain a lipid component consisting of an amino lipid, an auxiliary lipid, and a PEG lipid; and a nucleic acid component consisting of an mRNA encoding a Cas nuclease and a gRNA. In some compositions containing mRNA encoding a Cas nuclease and gRNA, the accessory lipid is cholesterol. In some compositions containing mRNA encoding a Cas nuclease and gRNA, the neutral lipid is DSPC. Some compositions containing mRNA encoding a Cas nuclease and gRNA contain less than about 1 mol% neutral lipid, such as neutral phospholipid. Some compositions containing mRNA encoding a Cas nuclease and gRNA contain less than about 0.5 mol% neutral lipid, such as neutral phospholipid. In some compositions, the LNP does not contain a neutral lipid, such as a neutral phospholipid. In further embodiments comprising mRNA encoding a Cas nuclease and gRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the aminolipid is selected from Lipid A and its equivalents, such as acetal analogues of Lipid A.

In certain embodiments, the LNP compositions comprise Cas nuclease mRNA, such as class 2 Cas mRNA, and at least one gRNA. In certain embodiments, the LNP composition has a ratio of gRNA to Cas nuclease mRNA, such as class 2 Cas nuclease mRNA, from about 25:1 to about 1:25. In certain embodiments, the LNP composition has a ratio of gRNA to Cas nuclease mRNA, such as class 2 Cas nuclease mRNA, from about 10:1 to about 1:10. In certain embodiments, the LNP composition has a gRNA to Cas nuclease mRNA, such as class 2 Cas nuclease mRNA, ratio of from about 8:1 to about 1:8. The ratios measured herein are by weight. In some embodiments, the LNP composition has a gRNA to Cas nuclease mRNA, such as class 2 Cas mRNA, ratio of from about 5:1 to about 1:5. In some embodiments, the ratio range is from about 3:1 to 1:3, from about 2:1 to 1:2, from about 5:1 to 1:2, from about 5:1 to 1:1, from about 3 :1 to 1:2, about 3:1 to 1:1, about 3:1, about 2:1 to 1:1. In some embodiments, the gRNA to mRNA ratio is about 3:1 or about 2:1. In some embodiments, the gRNA to mRNA ratio of a Cas nuclease, such as a class 2 Cas nuclease, is about 1:1. The ratio can be around 25:1, 10:1.5:1.3:1, 1:1, 1:3, 1:5, 1:10 or 1:25.

The LNP compositions described herein may contain a template nucleic acid. The template nucleic acid can be co-formulated with an mRNA encoding a Casnuclease, such as a class 2 Cas nuclease mRNA. In some embodiments, the template nucleic acid can be co-formulated with a guide RNA. In some embodiments, the template nucleic acid may be combined with both an mRNA encoding a Cas nuclease and a guide RNA. In some embodiments, the template nucleic acid may be composed separately from the Cas nuclease-encoding mRNA or guide RNA. The template nucleic acid may be delivered together or separately from the LNP compositions. In some

- 27 047139 embodiments, the template nucleic acid can be single-stranded or double-stranded, depending on the desired repair mechanism. The template may have regions of homology to the target DNA or to sequences adjacent to the target DNA.

In some embodiments, LNP is prepared by mixing an aqueous solution of RNA with a lipid solution based on an organic solvent, such as 100% ethanol. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. A pharmaceutically acceptable buffer may be used, for example, to administer LNP in vivo. In certain embodiments, the buffer is used to maintain the pH of the LNP-containing composition at pH 6.5 or higher. In certain embodiments, the buffer is used to maintain the pH of the LNP-containing composition at pH 7.0 or higher. In certain embodiments, the composition has a pH in the range of about 7.2 to about 7.7. In additional embodiments, the composition has a pH in the range of about 7.3 to about 7.7, or in the range of about 7.4 to about 7.6. In other embodiments, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH value of the composition can be measured using a pH microdetector. In certain embodiments, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO and ethylene glycol. Exemplary compositions may contain up to 10% cryoprotectant, such as, for example, sucrose. In certain embodiments, the LNP composition may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant. In certain embodiments, the LNP composition may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% sucrose. In some embodiments, the LNP composition may contain a buffer. In some embodiments, the buffer may comprise phosphate buffer (PBS), Tris buffer, citrate buffer, or mixtures thereof. In some illustrative embodiments, the buffer contains NaCl. In certain embodiments, NaCl is not used. Exemplary amounts of NaCl may range from about 20 to about 45 mM. Exemplary amounts of NaCl may range from about 40 to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer. Exemplary amounts of Tris can range from about 20 mM to about 60 mM. Exemplary amounts of Tris can range from about 40 to about 60 mM. In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer contains NaCl and Tris. Some exemplary embodiments of LNP compositions contain 5% sucrose and 45 mM NaCl in Tris buffer. In other illustrative embodiments, the compositions contain sucrose at about 5% w/v, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The amount of salt, buffer and cryoprotectant can be varied to maintain the osmolality of the entire formulation. For example, final osmolality may be maintained at less than 450 mOsm/L. In other embodiments, the osmolality is between 350 and 250 mOsm/L. Some embodiments have a final osmolality of 300 +/- 20 mOsm/L.

In some embodiments, microfluidic mixing, T-mixing, or cross-mixing is used. In certain aspects, flow rates, junction size, junction geometry, junction shape, tubing diameter, solutions and/or concentrations of RNA and lipids may be varied. LNP or LNP compositions can be concentrated or purified, for example, by dialysis, tangential flow filtration or chromatography. LNPs can be stored, for example, in the form of a suspension, emulsion or lyophilized powder. In some embodiments, the LNP composition is stored at 2-8°C; in certain aspects, the LNP composition is stored at room temperature. In additional embodiments, the LNP composition is stored frozen, for example at -20 or -80°C. In other embodiments, the LNP composition is stored at a temperature ranging from about 0 to about -80°C. Frozen LNP compositions can be thawed before use, eg on ice, at room temperature or at 25°C.

LNPs may be, for example, microspherical particles (including single- and multilayered vesicles, such as liposomes—lipid bilayers with a lamellar phase that, in some embodiments, are substantially spherical and, in more specific embodiments, may contain an aqueous core, e.g. , containing a significant portion of RNA molecules), a dispersed phase in an emulsion, micelles or a dispersed phase in a suspension.

Moreover, LNP compositions are biodegradable because they do not accumulate to cytotoxic levels in vivo at a therapeutically effective dose. In some embodiments, LNP compositions do not induce an innate immune response that results in significant side effects at the therapeutic dose level. In some embodiments, the LNP compositions provided herein do not cause toxicity at the therapeutic dose level.

In some embodiments, the pdi value may range from about 0.005 to about 0.75. In some embodiments, pdi may range from about 0.01 to

- 28 047139 about 0.5. In some embodiments, pdi may range from about zero to about 0.4. In some embodiments, pdi may range from about zero to about 0.35. In some embodiments, pdi may range from about zero to about 0.35. In some embodiments, pdi may range from about zero to about 0.3. In some embodiments, pdi may range from about zero to about 0.25. In some embodiments, pdi may range from about zero to about 0.2. In some embodiments, pdi may be less than about 0.08, 0.1, 0.15, 0.2, or 0.4.

The LNPs described herein have a size (eg, Z-average diameter) of about 1 to about 250 nm. In some embodiments, the LNPs have a size of from about 10 to about 200 nm. In additional embodiments, the LNPs have a size of from about 20 nm to about 150 nm. In some embodiments, the LNPs have a size of from about 50 nm to about 150 nm. In some embodiments, the LNPs have a size of from about 50 to about 100 nm. In some embodiments, the LNPs have a size of from about 50 nm to about 120 nm. In some embodiments, the LNPs have a size of from about 60 to about 100 nm. In some embodiments, the LNPs have a size of from about 75 nm to about 150 nm. In some embodiments, the LNPs have a size of from about 75 nm to about 120 nm. In some embodiments, the LNPs have a size of from about 75 to about 100 nm. Unless otherwise stated, all sizes mentioned herein are the average sizes (diameters) of fully formed nanoparticles measured by dynamic light scattering on a Malvern Zetasizer. The nanoparticle sample is diluted in phosphate-buffered saline (PBS) so that the intensity is approximately 200-400 kilopulses per second. Data are presented as intensity-weighted average (Z-mean diameter).

In some embodiments, LNPs are formed with an average encapsulation efficiency ranging from about 50 to about 100%. In some embodiments, LNPs are formed with an average encapsulation efficiency ranging from about 50 to about 70%. In some embodiments, LNPs are formed with an average encapsulation efficiency ranging from about 70 to about 90%. In some embodiments, LNPs are formed with an average encapsulation efficiency ranging from about 90 to about 100%. In some embodiments, LNPs are formed with an average encapsulation efficiency ranging from about 75 to about 95%.

In some embodiments, LNPs are formed with an average molecular weight ranging from about 1.00E+05 g/mol to about 1.00E+10 g/mol. In some embodiments, LNPs are formed with an average molecular weight ranging from about 5.00E+05 g/mol to about 7.00E+07 g/mol. In some embodiments, LNPs are formed with an average molecular weight ranging from about 1.00E+06 g/mol to about 1.00E+10 g/mol. In some embodiments, LNPs are formed with an average molecular weight ranging from about 1.00E+07 g/mol to about 1.00E+09 g/mol. In some embodiments, LNPs are formed with an average molecular weight ranging from about 5.00E+06 g/mol to about 5.00E+09 g/mol.

In some embodiments, the polydispersity (Mw/Mn; the ratio of mass average molar mass (Mw) to number average molar mass (Mn)) can be from about 1,000 to about 2,000. In some embodiments, Mw/Mn may range from about 1.00 to about 1.500. In some embodiments, Mw/Mn may range from about 1.020 to about 1.400. In some embodiments, Mw/Mn may range from about 1.010 to about 1.100. In some embodiments, Mw/Mn may range from about 1.100 to about 1.350.

Methods for constructing cells; engineered cells.

The LNP compositions described herein can be used in cell engineering methods through gene editing, both in vivo and in vitro. In some embodiments, the methods include contacting a cell with an LNP composition described herein.

In some embodiments, the methods include contacting a cell with a subject, such as a mammal, such as a human. In some embodiments, the cell is located in an organ, such as a liver, such as a mammalian liver, such as a human liver. In some embodiments, the cell is a liver cell, such as a mammalian liver cell, such as a human liver cell. In some embodiments, the cell is a hepatocyte, such as a mammalian hepatocyte, such as a human hepatocyte. In some embodiments, the liver cell is a stem cell. In some embodiments, the human liver cell may be a liver sinusoidal endothelial cell (LSEC). In some embodiments, the human liver cell may be a Kupffer cell. In some embodiments, the human liver cell may be a hepatic stellate cell. In some embodiments, the human liver cell may be a tumor cell. In some embodiments, the human liver cell may be a liver stem cell. In additional options

- 29 047139 implementation, the cell contains ApoE-binding receptors. In some embodiments, a liver cell, such as a hepatocyte, is in situ. In some embodiments, a liver cell, such as a hepatocyte, is isolated, for example, in culture, such as in a primary culture. Also provided are methods consistent with the applications disclosed herein, which include administering the LNP compositions described herein to a subject or contacting a cell, such as those described above, with the LNP compositions described herein.

In some embodiments, engineered cells are provided, such as an engineered cell derived from any of the cell types in the previous paragraph. Such engineered cells are obtained in accordance with the methods described herein. In some embodiments, the engineered cell is located within a tissue or organ, such as a liver, in a subject.

In some of the methods and cells described herein, the cell contains a modification, such as an insertion or deletion (indel), or substitution of nucleotides in the target sequence. In some embodiments, the modification comprises the insertion of 1, 2, 3, 4, or 5 or more nucleotides into the target sequence. In some embodiments, the modification comprises the insertion of 1 or 2 nucleotides into the target sequence. In other embodiments, the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a deletion of 1 or 2 nucleotides in the target sequence. In some embodiments, the modification contains an indel that results in a frameshift mutation in the target sequence. In some embodiments, the modification comprises replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises replacing 1 or 2 nucleotides in the target sequence. In some embodiments, the modification comprises one or more of an insertion, deletion, or substitution of nucleotides resulting from the inclusion of a template nucleic acid, such as any of the template nucleic acids described herein.

In some embodiments, a population of cells comprising engineered cells is provided, such as a population of cells comprising cells engineered in accordance with the methods described herein. In some embodiments, the population comprises engineered cells that are cultured in vitro. In some embodiments, the population is located within a tissue or organ, such as a liver, in a subject. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the cells in the population are engineered. In certain embodiments, the method described herein provides at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, or at least 95% editing efficiency (or editing percentage) as determined by indel detection. In other embodiments, the method described herein provides at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, or at least 95% DNA modification efficiency, as determined by detecting a change in sequence, whether insertion, deletion, substitution, or otherwise. In certain embodiments, the method described herein results in an editing efficiency level or DNA modification efficiency level of from about 5 to about 100%, from about 10 to about 50%, from about 20 to about 100%, from about 20 to about 80%, about 40 to about 100%, or about 40 to about 80% in a cell population.

In some of the methods and cells described herein, the cells in the population contain a modification, such as an indel or substitution, in the target sequence. In some embodiments, the modification comprises the insertion of 1, 2, 3, 4, or 5 or more nucleotides into the target sequence. In some embodiments, the modification comprises the insertion of 1 or 2 nucleotides into the target sequence. In other embodiments, the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a deletion of 1 or 2 nucleotides in the target sequence. In some embodiments, the modification results in a frameshift mutation in the target sequence. In some embodiments, modification of the content

- 30 047139 live indel, which leads to a mutation with a reading frame shift in the target sequence. In some embodiments, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 %, at least 96%, at least 97%, at least 98%, or at least 99% or more of the engineered cells in the population contain a frameshift mutation. In some embodiments, the modification comprises replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises replacing 1 or 2 nucleotides in the target sequence. In some embodiments, the modification comprises one or more of an insertion, deletion, or substitution of nucleotides resulting from the inclusion of a template nucleic acid, such as any of the template nucleic acids described herein.

Gene editing methods.

The LNP compositions described herein can be used for gene editing in vivo and in vitro. In one embodiment, one or more LNP compositions described herein can be administered to a subject in need thereof. In one embodiment, one or more LNP compositions described herein can be brought into contact with a cell. In one embodiment, a therapeutically effective amount of a composition described herein can be brought into contact with a cell of a subject in need thereof. In one embodiment, a genetically engineered cell can be produced by contacting the cell with an LNP composition described herein. In various embodiments, the methods include introducing a template nucleic acid into a cell or subject, as set forth above.

In some embodiments, the methods include administering the LNP composition to a cell associated with liver disease. In some embodiments, the methods include treating liver disease. In certain embodiments, the methods include contacting a liver cell with the LNP composition. In certain embodiments, the methods include contacting hepatocytes with the LNP composition. In some embodiments, the methods include contacting an ApoE-binding cell with the LNP composition.

In one embodiment, an LNP composition comprising mRNA encoding a class 2 Casnuclease and gRNA can be introduced into a cell, such as an ApoE-binding cell. In additional embodiments, the template nucleic acid is also introduced into the cell. In some cases, an LNP composition containing a class 2 Cas nuclease and ssRNA can be introduced into a cell, such as an ApoE-binding cell. In one embodiment, an LNP composition comprising mRNA encoding a class 2 Cas nuclease, a gRNA, and a template can be introduced into a cell. In some cases, an LNP composition containing Cas nuclease and sRNA can be introduced into a liver cell. In some cases, the liver cell is found in the subject.

In certain embodiments, the subject may receive a single dose of the LNP composition. In other examples, the subject may receive multiple doses of the LNP composition. In some embodiments, the LNP composition is administered 2-5 times. When more than one dose is administered, the doses may be administered at intervals of about 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days; at intervals of about 2, 3, 4, 5 or 6 months; or at intervals of about 1, 2, 3, 4 or 5 years. In some embodiments, editing improves after reintroduction of the LNP composition.

In one embodiment, an LNP composition containing mRNA encoding a Casnuclease, such as a class 2 Cas nuclease, can be introduced into a cell separately from the administration of the gRNA-containing composition. In one embodiment, an LNP composition comprising an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, and a gRNA can be introduced into a cell separately from the introduction of the template nucleic acid into the cell. In one embodiment, an LNP composition containing mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, can be introduced into a cell, followed by sequential administration of an LNP composition containing gRNA, and then introduction of the matrix into the cell. In embodiments in which the LNP composition containing the mRNA encoding the Cas nuclease is administered before the LNP composition containing the gRNA, the administrations may be separated by about 4, 6, 8, 12, or 24 hours; or 2, 3, 4, 5, 6 or 7 days.

In one embodiment, LNP compositions can be used to edit a gene, resulting in gene knockout. In one embodiment, LNP compositions can be used for gene editing, resulting in gene knockdown in a population of cells. In another embodiment, LNP compositions can be used for gene editing, resulting in gene correction. In an additional embodiment, LNP compositions can be used to edit a cell, resulting in gene insertion.

In one embodiment, administration of LNP compositions may result in gene editing that results in a durable response. For example, administration may result in a duration of response of a day, a month, a year, or more. As used herein, the term response duration means that after the cells have been edited using the method described herein

- 31 047139 document of the LNP composition, the resulting modification is still present for a certain period of time after administration of the LNP composition. The modification can be detected by measuring levels of the target protein. The modification can be detected by detecting the target DNA. In some embodiments, the duration of response may be at least 1 week. In other embodiments, the duration of response may be at least 2 weeks. In one embodiment, the duration of response may be at least 1 month. In some embodiments, the duration of response may be at least 2 months. In one embodiment, the duration of response may be at least 4 months. In one embodiment, the duration of response may be at least 6 months. In certain embodiments, the duration of response may be about 26 weeks. In some embodiments, the duration of response may be at least 1 year. In some embodiments, the duration of response may be at least 5 years. In some embodiments, the duration of the response may be at least 10 years. In some embodiments, a durable response is detected after at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 21, or 24 months, or by by measuring target protein levels or by detecting target DNA. In some embodiments, a sustained response is detected after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 years, either by measuring levels of the target protein or by detection of target DNA.

LNP compositions can be administered parenterally. LNP compositions can be administered directly into the bloodstream, tissue, muscle, or internal organ. Administration may be systemic, for example by injection or infusion. Application may be local. Suitable routes for administration include intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, subretinal, intravitreal, anterior chamber, intramuscular, intrasynovial, intradermal, and subcutaneous. Suitable delivery devices include needle (including microneedle) injectors, needleless injectors, osmotic pumps and infusion devices.

LNP compositions are typically, but not necessarily, administered as a formulation in combination with one or more pharmaceutically acceptable excipients. The term excipient includes any ingredient other than the compound(s) herein described, other lipid component(s) and biologically active agent. The excipient may provide functional (eg, drug release rate control) and/or non-functional (eg, processing aid or diluent) characteristics to the formulations. The choice of excipient will largely depend on factors such as the specific route of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

Parenteral formulations are usually aqueous or oily solutions or suspensions. When the formulation is aqueous, excipients such as sugars (including, but not limited to, glucose, mannitol, sorbitol, etc.), salts, carbohydrates and buffering agents (preferably for pH 3 to 9), but for some applications they may more suitably be formulated in a sterile non-aqueous solution or in a dried form for use in combination with a suitable carrier such as sterile pyrogen-free water (WFI).

Although the invention has been described in connection with the illustrated embodiments, it is understood that they are not intended to limit the invention to these embodiments. Rather, the invention is intended to cover all alternatives, modifications and equivalents, including equivalents of specific features, that may be included in the invention as defined in the appended claims.

Both the foregoing general description and the detailed description, as well as the following examples, are illustrative and explanatory only and do not limit the description. The section headings used herein are for organizational purposes only and should not be construed as limiting the intended subject matter of the invention in any way. In the event that any literature incorporated by reference conflicts with any term defined herein, the term given herein controls. All ranges reported in mol% cover endpoints unless otherwise noted.

It should be noted that, as used in this invention, the singular forms include a reference to the plural unless the context clearly indicates otherwise. Thus, for example, a reference to a composition includes a plurality of compositions, and a reference to a cell includes a plurality of cells and the like. The use of or is inclusive and means and/or unless otherwise specified.

Numeric ranges include numbers that define a range. Measured and measurable values are considered approximate, taking into account significant figures and error due to measurement. The term about or approximately means an acceptable error for a particular value, as determined by one skilled in the art, which depends in part on how the error is measured or determined.

- 32 047139 is the value. Using a modifier such as about before a range or before a list of values modifies each endpoint of the range or each value in the list. Around also includes a value or endpoint. For example, about 50-55 covers from about 50 to about 55. In addition, the uses of contain, contains, containing, consist of, consists of, consisting of, include, comprises, and including are not limiting.

Unless specifically stated in the above description, embodiments in the description that are referred to as containing various components are also considered to consist of or consist essentially of said components; embodiments in the description that are referred to as consisting of various components are also considered to contain or consist essentially of the listed components; embodiments in the specification that refer to various components are also considered as listed components; and embodiments in the specification that mention words consisting essentially of various components are also considered to be composed of or containing the listed components (this interchangeability does not apply to the use of these terms in the claims).

Examples

Example 1: LNP compositions for in vivo editing in mice.

Various LNP compositions have been prepared on a small scale to investigate their properties. In mouse liver percent editing assays, Cas9 mRNA and chemically modified sRNA targeting the mouse TTR sequence were formulated as LNPs with different mol% PEG, mol% Lipid A, and N:P ratios as described in Table 1 . 2 below.

Table 2 ________________________LNP compositions________________________

LNP No. %mol. Lipid A %mol. PEG-DMG N:P ratio (various) 45 2, 2.5, 3, 4, 5 4.5 (various) 45 2, 2.5, 3, 4, 5 6 (various) 50 2, 2.5, 3, 4, 5 4.5 (various) 50 2, 2.5, 3, 4, 5 6 (various) 55 2, 2.5, 3, 4, 5 4.5 (various) 55 2, 2.5, 3, 4, 5 6

In fig. 1, LNP formulations are identified on the x-axis based on their Lipid A mol% and N:P ratios, labeled as %CL; N:P. As indicated in the legend to FIG. 1, concentrations of 2, 2.5, 3, 4, or 5 mol% PEG2k-DMG were formulated with (1) 45 mol% Lipid A; 4.5 N:P (45; 4.5); (2) 45 mol% Lipid A; 6 N:P (45; 6); (3) 50 mol% Lipid A; 4.5 N:P (50; 4.5); (4) 50 mol% Lipid A; 6 N:P (50; 6); (5) 55 mol.% Lipid A; 4.5 N:P (55; 4.5); and (6) 55 mol% Lipid A; 6 N:P (55; 6). mol% DSPC was kept constant at 9 mol%, and mol% cholesterol was added to bring the balance of the lipid component of each formulation to 100 mol%. Each of the 30 formulations was formulated as described below and administered as a single dose of 1 mg per kg or 0.5 mg per kg doses of total RNA (Fig. 1A and Fig. 1B, respectively).

Composition of LNP - NanoAssemblr.

The lipid nanoparticle components were dissolved in 100% ethanol with the lipid component molar ratios given above. RNA cargo was dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in an RNA cargo concentration of approximately 0.45 mg/ml. LNPs were formulated with an aminolipid to RNA phosphate (N:P) molar ratio of about 4.5 or about 6, with an mRNA to gRNA mass ratio of 1:1.

LNPs were prepared by microfluidic mixing of lipid and RNA solutions using a Precision Nanosystems NanoAssemblr™ benchtop instrument according to the manufacturer's protocol. A 2:1 ratio of aqueous to organic solvent was maintained during mixing using differential flow rates. After mixing, LNPs were collected, diluted with water (approximately 1:1 v/v), incubated for 1 hour at room temperature, and further diluted with water (approximately 1:1 v/v) before the final buffer exchange. A final buffer exchange of 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS) was completed using PD-10 desalting columns (GE). When necessary, compounds were concentrated by centrifugation using Amicon 100 kDa cutoff centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNP was stored at 80°C until further use.

Analysis of compositions.

Dynamic light scattering (DLS) is used to determine the polydispersity index

- 33 047139 (pdi) and LNP size according to this description. DLS measures the light scattering that occurs when a sample is exposed to a light source. PDI, as determined from DLS measurements, is the distribution of particle size (relative to mean particle size) in a population, with a perfectly homogeneous population having a PDI of zero.

Electrophetic light scattering is used to determine the surface charge of LNPs at a given pH. Surface charge, or zeta potential, is a measure of the magnitude of electrostatic repulsion/attraction between particles in an LNP suspension.

Asymmetric Flow Field Fractionation - Multi-Angle Light Scattering (AF4MALS) is used to separate particles in a formulation by hydrodynamic radius and then subsequently measure the molecular weights, hydrodynamic radii, and rms radii of the fractionated particles. This allows the molecular weight and particle size distributions to be assessed, as well as secondary characteristics such as the Burchard-Stockmeier plot (the ratio of the root mean square (RMS) radius and the hydrodynamic radius over time, which indicates the internal core density of the particle) and the conformation map (logarithm of the RMS depending on the logarithm of molecular weight, where the slope of the resulting linear approximation gives the degree of compactness as a function of elongation).

Nanoparticle trajectory analysis (NTA, Malvern Nanosight) can be used to determine the particle size distribution of a formulation as well as the particle concentration. LNP samples are diluted appropriately and applied to a slide. The camera detects scattered light as particles slowly diffuse across the field of view. After recording the video, nanoparticle trajectory analysis processes the video by tracking the pixels and calculating the diffusion coefficient. This diffusion coefficient can be converted to the hydrodynamic radius of the particle. The instrument also counts the number of individual particles counted in the assay to determine the particle concentration.

Cryo-electron microscopy (cryo-EM) can be used to determine the particle size, morphology and structural characteristics of LNPs.

Analysis of the lipid composition of LNPs can be performed using liquid chromatography followed by charged aerosol detection (LC-CAD). This analysis can provide a comparison of actual lipid content with theoretical lipid content.

LNP formulations were analyzed for average particle size, polydispersity index (pdi), total RNA content, RNA encapsulation efficiency, and zeta potential. LNP formulations can be further characterized by lipid analysis, AF4-MALS, NTA and/or cryo-EM. Average particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS meter. LNP samples were diluted 30-fold in PBS before measurement by DLS. Along with the number average diameter and pdi, the Z-average diameter was given, which is an intensity-based measure of the average particle size. The Malvern Zetasizer is also used to measure the zeta potential of LNP. Samples are diluted 1:17 (50 µl per 800 µl) in ODC PBS, pH 7.4 before measurement.

A fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) is used to determine the concentration of total RNA and free RNA. Encapsulation efficiency is calculated as (Total RNA - Free RNA)/Total RNA. LNP samples are diluted appropriately with 1x TE buffer containing 0.2% Triton-X 100 for total RNA determination or 1x TE buffer for free RNA determination. Standard curves are prepared using the RNA stock solution used for formulation and diluted in 1x TE buffer +/- 0.2% Triton-X 100. Diluted RiboGreen® dye (according to the manufacturer's instructions) is then added to each of the standards and samples and leave to incubate for approximately 10 minutes at room temperature in the absence of light. A SpectraMax M5 microplate reader (Molecular Devices) is used to read samples with excitation, cutoff, and emission wavelengths of 488 nm, 515 nm, and 525 nm, respectively. Total RNA and free RNA were determined from the corresponding standard curves.

Encapsulation efficiency is calculated as (Total RNA - Free RNA)/Total RNA. The same procedure can be used to determine the encapsulation efficiency of a DNA or cargo-containing nucleic acid component. For single-stranded DNA, you can use Oligreen dye, and for double-stranded DNA, you can use PicoGreen dye.

AF4-MALS is used to determine molecular weight and particle size distributions and secondary statistics from these calculations. LNPs are appropriately diluted and injected into the AF4 separation channel using an HPLC autosampler, where they are focused and then eluted along an exponential gradient in a cross-flow through the channel. All liquid is driven by an HPLC pump and a Wyatt Eclipse instrument. Particles eluting from the AF4 channel pass through a UV detector, a multi-angle laser light scattering detector, a quasi-elastic light scattering detector, and a differential refractometric detector. Not

- 34 047139 the processed data is processed using the Debye model to determine the molecular mass and RMS radius from the detector signals.

Lipid components in LNPs are quantitatively analyzed using HPLC coupled with a charged aerosol detector (CAD). Chromatographic separation of the 4 lipid components is achieved using reverse phase HPLC. CAD is a destructive mass detector that detects all non-volatile compounds and the signal is constant regardless of the structure of the analyte.

Cargo mRNA Cas9 and gRNA.

Cargo Cas9 mRNA was produced by in vitro transcription. Capped and polyadenylated Cas9 mRNA containing 1X NLS (SEQ ID NO: 48) was produced by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing the T7 promoter and a 100-nucleotide poly(A/T) region was linearized by incubation at 37°C for 2 hours with XbaI under the following conditions: 200 ng/μl plasmid, 2 U/μl XbaI (NEB) , and 1x reaction buffer. XbaI was inactivated by heating the reaction mixture at 65°C for 20 minutes. The linearized plasmid was purified from enzyme and buffer salts using a Maxi Spin silica spin column (Epoch Life Sciences) and analyzed on an agarose gel to confirm linearization. The IVT reaction mixture to obtain modified Cas9 mRNA was incubated at 37°C for 4 hours under the following conditions: 50 ng/μl linearized plasmid; 2 mM each of GTP, ATP, CTP, and N-methyl pseudo-UTP (Trilink); 10 mM ARCA (Trilink); 5 units/μl T7 RNA polymerase (NEB); 1 U/μl murine RNase inhibitor (NEB); 0.004 U/μL inorganic E. coli pyrophosphatase (NEB); and 1x reaction buffer. After a 4-hour incubation, TURBO DNase (ThermoFisher) was added to a final concentration of 0.01 U/μl and the reaction mixture was incubated for an additional 30 minutes to remove template DNA. Cas9 mRNA was purified from enzyme and nucleotides using the MegaClear Transcription Clean-up kit according to the manufacturer's protocol (ThermoFisher). Alternatively, Cas9 mRNA was purified by LiCl precipitation.

In this example, the ssRNA was chemically synthesized and obtained from a commercial supplier. The sequence of sg282 is shown below with 2'-O-methyl modifications and phosphorothioate linkages as follows (m=2'-OMe; * = phosphorothioate):

mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmAmA mUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAm AmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*m U*mU.

(SEQ ID NO:42).

LNP

The final LNPs were characterized to determine encapsulation efficiency, polydispersity index, and average particle size according to the analytical methods presented above.

LNP was administered to mice (single dose of 1 mg/kg or 0.5 mg/kg) and genomic DNA was isolated for next generation sequencing (NGS) analysis as described below.

In vivo delivery of LNP.

Female CD-1 mice between 6 and 10 weeks of age were used in each study. Animals were weighed and grouped according to body weight to prepare dosing solutions based on group average weight. LNP was administered through the lateral tail vein in a volume of 0.2 ml per animal (approximately 10 ml per kilogram of body weight). Animals were monitored for adverse events approximately 6 hours after dosing. Body weight was measured twenty-four hours after administration, and the animals were euthanized at various time points by exsanguination by cardiac puncture under isoflurane anesthesia. Blood was collected into serum separation tubes or into tubes containing buffered sodium citrate solution to obtain plasma as described herein. For studies involving in vivo editing, liver tissue was collected from the middle lobe or from three independent lobes (eg, right medial, left medial, and left lateral lobes) of each animal for DNA extraction and analysis.

Cohorts of mice were examined for hepatic editing by next generation sequencing (NGS) and serum TTR levels (data not shown).

Analysis of transthyretin (TTR) by ELISA.

Blood was collected and serum isolated as indicated. Total TTR levels in mouse serum were determined using a mouse prealbumin (transthyretin) ELISA kit (Aviva Systems Biology, cat. OKIA00111). TTR levels in rat serum were measured using a rat-specific ELISA kit (Aviva Systems Biology, catalog number OKIA00159) according to the manufacturing protocol. Briefly, sera were serially diluted with sample diluent from the kit to a final dilution of 10,000-fold. This diluted sample was then added to the ELISA plates and the assay was then performed as directed.

NGS sequencing.

Briefly, to quantify the effectiveness of editing in a target location

- 35 047139 genomic DNA was isolated from the genome and deep sequencing was used to detect the presence of insertions and deletions introduced by gene editing.

Primers for PTR were designed around the target site (eg, TTR), and the genomic region of interest was amplified. The primer sequences are given below. Additional PCR was performed according to the manufacturer's protocols (Illumina) to add the necessary chemical components for sequencing. Amplicons were sequenced on an Illumina MiSeq instrument. Reads were aligned to the human reference genome (e.g. hg38) after excluding those with low quality scores. The resulting files containing reads were mapped to a reference genome (BAM files), where reads that overlapped the target region of interest were selected, and the number of wild-type reads versus the number of reads that contained an insertion, substitution, or deletion was calculated.

Percentage editing (e.g., editing efficiency or percent editing) is defined as the total number of sequence reads with insertions or deletions relative to the total number of sequence reads, including wild type.

In fig. Figure 1 shows the percentage of editing in mouse liver measured by NGS. As shown in FIG. 1A, when 1 mg per kg RNA is administered, in vivo editing percentages range from about 20 to more than 60% of editing in the liver. At a dose of 0.5 mg per kg, Fig. 1B, editing in the liver from about 10 to 60% was observed. In this in vivo test in mice, all formulations efficiently delivered Cas9 mRNA and gRNA to liver cells, indicating high CRISPR/Cas nuclease activity at the target site as measured by NGS for each LNP formulation. LNPs containing 5% PEGlipid had lower encapsulation (data not shown) and slightly reduced efficacy.

Example 2 Analysis of LNP composition.

Analytical characterization of LNPs demonstrates improved physicochemical parameters for LNPs formulated with increasing amounts of Lipid A and PEG lipid. Compositions that contain 2 mol.% or 3 mol.% PEG lipid (PEG2k-DMG) are presented in table. 3 below.

Table 3

LNP898 LNP897 LNP966 LNP969 CL/cholesterol/DSPC/PEG (theoretical mol.%) 45/44/9/2 45/43/9/3 50/38/9/3 55/33/9/3 mRNA Cas9 SEQ GO NO:48 Cas9 SEQ ID NO:48 Cas9 U-dep SEQ ID NO:43 Cas9 U-dep SEQ ID NO:43 gRNA G502 SEQ ID NO:70 G502 SEQ ID NO:70 G534 SEQ ID NO:72 G534 SEQ ID NO:72 N/P 4.5 4.5 6.0 6.0

LNP composition - Cross flow.

LNPs were prepared by fluid impingement mixing of lipid in ethanol with two volumes of RNA solutions and one volume of water. The lipid in ethanol is mixed through a mixing unit with two volumes of RNA solution. The fourth water flow is mixed with the output flow of the unit through the T-shaped connection. (See WO 2016010840 in Fig. 2). LNP was maintained at room temperature for 1 hour and then further diluted with water (approximately 1:1 v/v). Diluted LNPs were concentrated using tangential flow filtration on a flat sheet cartridge (Sartorius, HOMM 100 kDa) and then buffer exchanged by diafiltration to 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7, 5 (TSS). Alternatively, the final buffer exchange to TSS was completed using PD-10 desalting columns (GE). When necessary, compounds were concentrated by centrifugation using Amicon 100 kDa cutoff centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNP was stored at 4°C or -80°C until further use.

Cas9 mRNA and sRNA were prepared as in Example 1, except that capped and polyadenylated U-depleted Cas9 mRNA (Cas9 Udep) contains SEQ ID N: 43. Sg282 is described in Example 1, and the sequence for sg534 (G534) is given below :

mA*mC*mG*CAAAUAUCAGUCCAGCGGUUUUAGAmGmCmUmAmGmAmAmA mUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAm AmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*m U*mU (SEQ ID NO:72)

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LNP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency as described in Example 1.

Analysis of average particle size, polydispersity (PDI), total RNA content and RNA encapsulation efficiency are shown in Table. 4. In addition to the theoretical lipid concentrations in the LNP compositions, lipid analysis demonstrated actual mol% lipid levels as listed in Table 1. 5 below.

Table 4

LNP INN N/P Z-mean (nm) PDI Average (nm) Conc. RNA (mg/ml) Encaps. (%) LNP898 4.5 87.91 0.030 71.33 1.53 98 LNP897 4.5 74.05 0.036 58.55 1.43 98 LNP966 6.0 82.78 0.010 67.86 2.12 98 LNP969 6.0 92.97 0.042 75.52 2.09 97

Table 5

LNP ID no. Lipid ratio (Lipid A/cholest./DSPC/PEG) (theoretical and actual) Lipid A, mg/ml (theoretical and actual) Chol ed., mg/ml (theoretical and actual) DSPC, mg/ml (theoretical and actual) PEG, mg/ml (theoretical and actual) LNP8 98 45/44/9/2 46.1/42.6/9.2/2 18.0 18.3 8.0 7.7 3.3 3.4 2.3 2.4 LNP8 97 45/43/9/3 44.8/42.9/9.2/3.1 18.0 17.8 7.8 7.7 3.3 3.4 3.5 3.6 LNP9 66 50/38/9/3 50.0/38.0/8.8/3.1 33.4 35.6 11.5 12.3 5.6 5.8 5.8 6.5 LNP9 69 55/33/9/3 54.8/33.2/8.8/3.2 33.4 31.6 9.1 8.7 5.1 4.7 5.3 5.4

To further analyze the physicochemical properties of LNP897, LNP898, LNP966 and LNP969 were subjected to asymmetric flow fractionation-multi-angle light scattering (AF4-MALS) analysis. The AF4-MALS instrument measures particle size and molecular weight distributions and provides information on particle conformation and density.

LNPs are injected into the AF4 separation channel using an HPLC autosampler, where they are focused and then eluted with an exponential gradient across the channel. All liquid is driven by an HPLC pump and a Wyatt Eclipse instrument. Particles eluting from channel AF4 pass through a UV detector, a Wyatt Heleos II multi-angle laser light scattering detector, a quasi-elastic light scattering detector, and a Wyatt Optilab T-rEX differential refractometric detector. The raw data is processed in Wyatt Astra 7 software using the Debye model to determine the molecular mass and RMS radius from the detector signals.

A plot of the logarithmic derivative of the molar mass for LNP is shown in Fig. 2A. Briefly, the X-axis indicates the molar mass (g/mol) and the Y-axis indicates the derivative of the mole fraction. A plot of the logarithmic derivative of molar mass shows the distribution of different molecular masses measured for a particular composition. This provides data on the molecular mass mode as well as the overall molecular mass distribution within the composition, which provides a better picture of particle heterogeneity than average molecular mass.

The heterogeneity of different LNP compositions is determined by measuring the molar mass in different

- 37 047139 moments and calculations of the ratio of the weighted average molar mass (Mw) to the number average molar mass (Mn) to obtain the polydispersity Mw/Mn. The polydispersity plot for these different formulations is presented in FIG. 2B.

The data shows a denser particle distribution with 3 mol% PEG and 50 and 55 mol% Lipid A at N/P 6.0, as shown in FIG. 2A. This is reflected in dense polydispersity, as shown in Fig. 2B.

Example 3: AF4 MALS Data - Additional Formulations.

Analytical characterization of LNPs demonstrates improved physicochemical parameters in LNPs formulated with increasing amounts of Lipid A. Compositions that contain either 45 mol%, 50 mol%, or 55 mol% Lipid A with two different gRNAs are presented in Table 1. 6 below.

Table 6

LNP1021 LNP1022 LNP1023 LNP1024 LNP1025 CL/cholesterol/DSPC/PEG (theoretical mol.%) 50/38/9/3 55/33/9/3 45/43/9/3 50/38/9/3 55/33/9/3 mRNA Cas9 Udep SEQID NO:43 Cas9 Udep SEQID NO:43 Cas9 Udep SEQID NO:43 Cas9 Udep SEQID NO:43 Cas9 Udep SEQID NO:43 gRNA G502 SEQID NO:70 G502 SEQID NO:70 G502 SEQID NO:70 G509 SEQID NO:71 G509 SEQID NO:71 N/P 6.0 6.0 4.5 6.0 6.0

LNPs were formulated as described in Example 2.

Cas9 mRNA and ssRNA were prepared as described above.

LNP compositions were characterized to determine encapsulation efficiency, polydispersity index, and average particle size as described in Example 1.

Analysis of the average particle size, polydispersity (PDI), total RNA content, and RNA encapsulation efficiency is given in Table. 7. In addition to the theoretical lipid concentrations in the LNP compositions, lipid analysis demonstrated actual mol% lipid levels as listed in Table 1. 8 below.

Table 7

LNP HH№ N/P Z-mean (nm) PDI Average (nm) Conc. RNA (mg/ml) Encaps. (%) LNP1021 6.0 83.18 0.027 67.15 1.63 98 LNP1022 6.0 94.08 0.005 78.28 1.60 97 LNP1023 4.5 74.01 0.017 61.11 1.61 97 LNP1024 6.0 85.37 0.002 70.42 1.59 97 LNP1025 6.0 94.47 0.018 77.71 1.60 98

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Table 8

LNP ID no. Lipid ratio (Lipid A/cholest./DSPC/PEG) (theoretical and actual) Lipid A, mg/ml (theoretical and actual) Chol ed., mg/ml (theoretical and actual) DSPC, mg/ml (theoretical and actual) PEG, mg/ml (theoretical and actual) LNP1 021 50/38/9/3 50.9/37.4/8.6/3, 1 23.6 21.6 8.1 7.2 3.9 3.4 4.1 3.8 LNP1 022 55/33/9/3 55.2/33.0/8.7/3, 1 23.6 20.4 6.4 5.5 3.6 3.0 3.7 3.4 LNP1 023 45/43/9/3 45.9/42.4/8.6/3, 1 17.7 15.3 7.7 6.4 3.3 2.7 3.4 3.0 LNP1 024 50/38/9/3 50.5/37.9/8.5/3, 0 23.6 22.4 8.1 7.6 3.9 3.5 4.1 3.9 LNP1 024 55/33/9/3 55.5/33.1/8.5/3, 0 23.6 21.3 6.4 5.8 3.6 3.0 3.7 3.4

To further analyze the physicochemical properties of LNP1021, LNP1022, LNP1023, LNP1024 and LNP1025 were subjected to asymmetric flow fractionation multi-angle light scattering (AF4-MALS) analysis. The AF4-MALS instrument measures particle size and molecular weight distributions and provides information on particle conformation and density.

LNPs were examined using AF4-MALS as described in Example 1.

A plot of the logarithmic derivative of the molar mass for LNP is shown in Fig. 3A. Briefly, the X-axis indicates the molar mass (g/mol) and the Y-axis indicates the derivative of the mole fraction. A plot of the logarithmic derivative of molar mass shows the distribution of different molecular masses calculated for a particular composition. This provides data on the molecular mass mode as well as the overall molecular mass distribution within the composition, which provides a better picture of particle heterogeneity than average molecular mass.

The average molecular weight is shown in Fig. 3B. The average molecular weight is the average of the entire distribution, but does not provide information about the shape of that distribution. LNP1022 and LNP1025 have the same average molecular weight, but LNP1022 has a slightly wider distribution.

The heterogeneity of different LNP compositions is calculated by determining the molar mass at various times and calculating the ratio of the weighted average molar mass (Mw) to the number average molar mass (Mn) to obtain the polydispersity Mw/Mn. The polydispersity plot for these different formulations is presented in FIG. 4A.

Additionally, the Burchard-Stockmeier plot for LNP compositions is presented in FIG. 4B. The Burchard-Stockmeier plot demonstrates the RMS of the radius to the hydrodynamic radius when the composition is eluted from the AF4 channel. This provides information about the internal density of lipid nanoparticles. In fig. 4B shows that LNP1021, LNP1022 and LNP1023 have different profiles in this dimension.

Example 4: Increased PEG lipid content maintains efficacy with decreased cytokine response.

In another study, PEG-DMG lipid was compared in LNP formulations containing 2 mol.% or 3 mol.% PEG-lipid. Compositions that contain either 2 mol.% or 3 mol.% PEG-DMG are presented in table. 9 below.

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Table 9

LNP809 LNP810 CL/cholesterol/DSPC/PEG (theoretical mol.%) 45/44/9/2 45/43/9/3 mRNA Cas9 SEQ ID NO:48 Cas9 SEQ ID NO:48 gRNA G390 G390 SEQ ID NO:69 SEQ ID NO:69 N/P 4.5 4.5

LNPs were prepared using the method described in Example 2. Cas9 mRNA and sRNA were prepared as in Example 1, with the sequence sg390 (G390) shown below:

mG*mC*mC*GAGUCUGGAGAGCUGCAGUUUUAGAmGmCmUmAmGmAmAmA mUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAm

AmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO:69).

LNP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency as described in Example 1.

Analysis of average particle size, polydispersity (PDI), total RNA content and RNA encapsulation efficiency are shown in Table. 10. In addition to the theoretical lipid concentrations in the LNP compositions, lipid analysis demonstrated actual mol% lipid levels as listed in Table 1. 11 below.

Table 10

LNP HH№ N/P Z-mean (nm) PDI Average (nm) Conc. RNA (mg/ml) Encaps. (%) LNP809 4.5 89.85 0.060 72.10 2.45 97 LNP810 4.5 75.26 0.025 61.17 2.14 97

Table 11

LNP ID no. Lipid ratio (Lipid A/cholest./DSPC/PEG) (theoretical and actual) Lipid A, mg/ml (theoretical and actual) Chol ed., mg/ml (theoretical and actual) DSPC, mg/ml (theoretical and actual) PEG, mg/ml (theoretical and actual) LNP8 09 45/44/9/2 45.7/43.3/9.0/2, 1 28.6 30.5 12.7 13.1 5.3 5.6 3.7 4.0 LNP8 10 45/43/9/3 45.0/42.3/9.7/3, 0 25.2 24.7 10.9 10.5 4.7 4.9 4.9 4.7

Rat serum cytokines were assessed using a multiplex assay using Luminex magnetic beads (Milliplex MAP magnetic bead assay from Millipore Sigma, catalog number RECYTMAG-65K) with MCP-1, IL-6, TNF-alpha, and IFN-gamma assays. Assay beads were read on a BioRad BioPlex-200, and cytokine concentrations were calculated from a standard curve using 4-parameter logistic regression with BioPlex Manager version 6.1 software. The data is presented in Fig. 5. See fig. 5A (serum TTR), FIG. 5B (liver editing) and FIG. 5C (cytokine p MCP1).

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TTR levels in rat serum were measured using a rat-specific ELISA kit (Aviva Systems Biology, catalog number OKIA00159) according to the manufacturing protocol. Briefly, sera were serially diluted with sample diluent from the kit to a final dilution of 10,000-fold. This diluted sample was then added to the ELISA plates and the assay was then performed as directed.

Genomic DNA was isolated from approximately 10 mg of liver tissue and analyzed using NGS as described above. The PCR primer sequences for amplification are described below.

In fig. 5A and FIG. 5B shows that TTR knockdown in serum and editing in liver were sufficient for the 2 mol% and 3 mol% PEG formulations. In FIG. 5C shows that the MCP-1 response is reduced using formulations with 3 mol.% PEG.

Example 5 Delivery of LNP to Non-Human Primates

Three studies were conducted with LNP formulations prepared as described in Example 1. Specific molar amounts and cargos are given in Tables 12-26. Each composition containing Cas9 mRNA and guide RNA (gRNA) had an mRNA:gRNA ratio of 1:1 by weight. Doses of LNP (in mg/kg, total RNA content), route of administration, and whether animals received pretreatment with dexamethasone are indicated in the tables. For animals pretreated with dexamethasone (Dex), Dex was administered at a dose of 2 mg/kg by intravenous bolus injection 1 hour before LNP or vehicle administration.

To analyze blood chemistry, blood was collected from animals at the time points indicated in the tables below for each factor measured. Cytokine induction was measured before and after NHP treatment. A minimum of 0.5 ml of whole blood was collected from the peripheral vein of immobilized, awake animals into a 4-ml serum separation tube. Blood was allowed to clot for at least 30 minutes at room temperature, followed by centrifugation at 2000 x g for 15 minutes. Serum was aliquoted into 2 polypropylene microtubes of 120 μl each and stored at -60 to -86°C until analysis. A custom non-human primate U-Plex Cytokine kit from Meso Scale Discovery (MSD) was used for the analysis. The following parameters were included in the analysis: IFN-gamma, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p40, MCP-1 and TNF-alpha, with special emphasis on IL-6 and MSR-1. Reagents and kit standards were prepared according to the instructions in the manufacturer's protocol. NHP serum was used in its pure form. The plates were read on an MSD Sector Imager 6000 and analyzed using MSD Discovery Workbench version 4012 software.

Complement levels were measured in animals before and after treatment using an enzyme-linked immunosorbent assay. Whole blood (0.5 ml) was collected from the peripheral vein of immobilized, awake animals into a tube containing 0.5 ml EDTA-K2. The blood was centrifuged at 2000 xg for 15 minutes. Plasma was aliquoted into 2 polypropylene microtubes of 120 μl each and stored at -60 to -86°C until analysis. The EIA Quidel MicroVue Complement Plus kit (C3a - cat. No. A031) or (Bb - Cat. No. A027) was used for analysis. Reagents and kit standards were prepared according to the instructions in the manufacturer's protocol. The plates were read on an MSD Sector Imager 6000 at an optical density of 450 nm. Results were analyzed using 4-variable logistic regression.

Data for cytokine induction and complement activation are presented in the tables below. BLQ means below the limit of quantitation.

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Table 12

Study 1

Treatment group Molar ratios (Lipid A, cholesterol, DSPC and PEG2cDMG, respectively N:P Cargo Volume M samples (η) Method of administration Dose level containing no total RNA (mg/kg) Dexa metaz he (1) TSS (carrier ) n/a n/a n/a 3 i.v. - infusion n/a No (2) LNP699 G502 45/44/9/2 4.5 mRNA Cas9 (SEQ ID NO:2); G000502 3 i.v. - infusion 3 No (3) LNP688 G506 45/44/9/2 4.5 mRNA Cas9 (SEQ ID NO:2); G000506 3 i.v. - infusion 3 No (4) LNP689 G509 45/44/9/2 4.5 mRNA Cas9 (SEQ ID NO:2); G000509 3 i.v. - infusion 3 No (5) LNP690 G510 45/44/9/2 4.5 mRNA Cas9 (SEQ ID NO:2); G000510 3 i.v. - infusion 3 No

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Table 13

Study 2

Treatment group Molar ratios (Lipid A, cholesterol, DSPC and PEG2kDMG, respectively N:P Cargo Sample size and (η) Method b introduced nya Dose level containing no total RNA (mg/kg) Dec same pelvis (1) TSS (carrier) n/a n/a 1 IV bolus n/a Yes (2) TSS (carrier) n/a n/a 1 IV bolus n/a No (3) LNP898 G502 45/44/9/2 4.5 mRNA Cas9 (SEQ ID NO:2); G000502 1 IV infusion 3 Yes (4) 45/44/9/2 4.5 mRNA 1 i.v. - 3 No

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LNP898 G502 Cas9 (SEQ ID NO:2); G000502 infusion I (5) LNP897 G502 45/43/9/3 4.5 mRNA Cas9 (SEQ ID NO:2); G000502 1 i.v. - bolus 3 Yes (6) LNP897 G502 45/43/9/3 4.5 mRNA Cas9 (SEQ ID NO:2); G000502 1 i.v. - bolus 3 No (7) LNP897 G502 45/43/9/3 4.5 mRNA Cas9 (SEQ ID NO:2); G000502 1 IV infusion 3 Yes (8) LNP897 G502 45/43/9/3 4.5 mRNA Cas9 (SEQ ID NO:2); G000502 1 IV infusion 3 No (9) LNP916 GFP 45/43/9/3 4.5 mRNA eGFP (SEQ ID NO:) 1 IV infusion 6 Yes (10) LNP916 GFP 45/43/9/3 4.5 mRNA eGFP (SEQ ID NO:) 1 IV infusion 6 No

Table 14

Study 3

Treatment group Molar ratios N:P Cargo Volume selection Way Dose level Dex amet

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(Lipid A, cholesterol, DSPC and PEG2k- DMG, respectively KII(n) enter Research Institute total RNA content (mg/kg) azon (l)TSS n/a n/a n/a 3 i/v pain With n/a No (2) LNP1021 G502 50/38/9/3 6 mRNA Cas9 (SEQ. ID NO:1); G00050 2 3 i/v pain With 1 No (3) LNP1021 G502 50/38/9/4 6 mRNA Cas9 (SEQ. ID NO:1); G00050 2 1 i/v pain with 1 Yes (4) LNP1022 G502 55/33/9/3 6 mRNA Cas9 (SEQ. ID NO:1); G00050 2 3 i/v pain With 1 No (5) LNP1023 G502 45/43/9/3 4.5 mRNA Cas9 (SEQ. ID NO:1); G00050 3 i/v pain With 3 No

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2 (6) LNP1024 G509 50/38/9/3 6 mRNA Cas9 (SEQ ID NO:1); G00050 9 3 b/b- I'm in pain c 1 No (7) LNP1024 G509 50/38/9/4 6 mRNA Cas9 (SEQ ID NO:1); G00050 9 1 b/b- I'm in pain c 1 Yes (8) LNP1025 G509 55/33/9/3 6 mRNA Cas9 (SEQ ID NO:1); G00050 9 3 b/b- I'm in pain c 1 No (9) LNP1021 G502 50/38/9/3 6 mRNA Cas9 (SEQ ID NO:1); G00050 2 1 b/b- I'm in pain c 3 No (10) LNP1022 G502 50/38/9/3 6 mRNA Cas9 (SEQ ID NO:1); G00050 1 b/b- I'm in pain c 3 No 2

Table 15

IL-6 measurements from study 1

Treatment group Preliminary blood sampling 6 hours 24 hours (1) TSS (carrier) 5.71±2.70 29.1±20.37 7.05±3.49 (2) LNP699 G502 9.73±8.34 1296.41±664.71 5.43±7.68 (3) LNP688 G506 16.83±4.08 1749.47± 1727.22 38.57±39.39 (4) LNP689 G509 18.11±11.51 1353.49±766.66 32.42±18.40 (5) LNP690 G510 13.95±1.85 11838=1=17161.74 90.07±96.02

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Table 16

MCP-1 measurements from Study 1

Treatment group Preliminary blood sampling 6 hours 24 hours (1)TSS (carrier) 810.49± 178.27 1351.16=1=397.31 745.25±56.49 (2) LNP699 G502 842.31±350.65 19298.49± 11981.14 2092.89± 171.21 (3) LNP688 G506 1190.79±383.64 13500.17±12691.60 1414.71±422.43 (4) LNP689 G509 838.63±284.42 14427.7±8715.48 1590±813.23 (5) LNP690 G510 785.32±108.97 52557.24±48034.68 6319.77±983.37

Table 17

Complement C3a measurements from Study 1

Treatment group Preliminary blood sampling 6 hours 7 days (1)TSS (carrier) 23.9±11.95 25.51±14.79 30.67±18.36 (2) LNP699 G502 32.36±11.29 94.33±58.45 38.50±12.69 (3) LNP688 G506 22.30±1.73 127.00±22.34 37.80±6.86 (4) LNP689 G509 35.83±21.94 174.00±44.51 50.83±21.92 (5) LNP690 G510 36.30±8.21 163.00±40.60 42.50± 12.44

Table 18

bb complement measurements from study 1

Treatment group 04-bb Preliminary blood sampling 6 hours 7 days (1)TSS (carrier) Control 1.53±0.19 3.37±2.13 1.43±0.71 (2) LNP699 G502 G502 1.45±0.39 9.01±5.28 1.57±0.54 (3) LNP688 G506 1.45±0.78 11.78±2.33 1.78±0.84 G506 (4) LNP689 G509 G509 1.95±0.99 15.73±2.23 2.83±0.88 (5) LNP690 G510 G510 2.12±0.44 13.57±1.23 2.21±0.72

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Table 19

IL-6 measurements from study 2

Treatment group Preliminary blood sampling 90 min 6 hours 24 hours 7 days (1)TSS (carrier) 1.77 11.46 4.2 2.76 3.01 (2)TSS (carrier) 5.23 18.11 20.36 13.2 6.36 (3) LNP898 G502 2.02 1305.75 1138.22 383.32 16.02 (4) LNP898 G502 2.34 37.19 91.59 14.11 3.07 (5) LNP897 G502 2.1 55.79 6.89 2.26 2.01 (6) LNP897 G502 6.8 10.1 44.72 5.4 2.01 (7)LNP897 G502 1.97 44.87 32.61 2.97 1.11 (8)LNP897 G502 3.14 37.68 73.41 8.58 2.22 (9) LNP916 GFP 1.6 BLQ 95.32 27.58 BLQ (10) LNP916 GFP 2.43 BLQ 883.01 66.71 BLQ

Table 20

MCP-1 measurements from Study 2

Treatment group Preliminary fence blood 90 min 6 hours 24 hours 7 days (1) TSS (carrier) 312.12 197.24 145.36 177.02 403.82

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(2) TSS (carrier) 232.44 175.08 187.72 136.64 325.69 (3) LNP898 G502 249.1 2183.5 1814.64 1887.41 372.38 (4) LNP898 G502 349.51 430.49 5635.55 953.05 236.6 (5) LNP897 G502 492.3 989.98 409.08 302.97 506.82 (6) LNP897 G502 283.79 225.1 1141.08 484.59 259.46 (7) LNP897 G502 223.16 349.79 398.57 172.67 287.09 (8) LNP897 G502 584.42 853.51 3880.81 1588.46 692.99 (9) LNP916 GFP 325.84 BLQ 1189.97 2279.82 BLQ (10) LNP916 GFP 175.47 BLQ 3284.16 2023.53 BLQ

Table 21

Complement C3a measurements from study 2

Treatment group Preliminary blood sampling 90 min 6 hours 24 hours 7 days (1)TSS (carrier) 0.087 0.096 0.048 0.033 0.038 (2)TSS (carrier) 0.369 0.311 0.146 0.1 0.106 (3) LNP898 G502 0.087 0.953 0.647 0.277 0.065 (4) LNP898 G502 0.099 0.262 0.123 0.049 0.044 (5) LNP897 G502 0.067 0.479 0.209 0.036 0.036 (6) LNP897 G502 0.141 0.433 0.34 0.11 0.074 (7) LNP897 G502 0.1 0.345 0.396 0.096 0.127 (8)LNP897 G502 0.261 0.458 0.409 0.244 0.313 (9) LNP916 GFP 0.149 BLQ 0.714 0.382 BLQ (10) LNP916 GFP 0.117 BLQ 0.752 0.723 BLQ

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Table 22

bb complement measurements from study 2

Treatment group Preliminary blood sampling 90 min 6 hours 24 hours 7 days (1)TSS (carrier) 0.087 0.096 0.048 0.033 0.038 (2)TSS (carrier) 0.369 0.311 0.146 0.1 0.106 (3) LNP898 G502 0.087 0.953 0.647 0.277 0.065 (4) LNP898 G502 0.099 0.262 0.123 0.049 0.044 (5) LNP897 G502 0.067 0.479 0.209 0.036 0.036 (6) LNP897 G502 0.141 0.433 0.34 0.11 0.074 (7)LNP897 G502 0.1 0.345 0.396 0.096 0.127 (8) LNP897 G502 0.261 0.458 0.409 0.244 0.313 (9) LNP916 GFP 0.149 BLQ 0.714 0.382 BLQ (10) LNP916 GFP 0.117 BLQ 0.752 0.723 BLQ

Table 23

IL-6 measurements from Study 3

Treatment group Preliminary blood sampling 90 min 6 hours 24 hours 7 days (l)TSS 1.89±0.97 2.56±1.41 0.90±0.71 BLQ 0.08 (2) LNP1021 G502 210±0.35 7.44±5.16 6.94±8.45 1.07±1.11 1.76±0.98 (3) LNP1021 G502 0.79 2.96 20.42±31.6 4.25 13.94±10.1 0.67 0.27 (4) LNP1022 G502 1.54±1.32 0 0 0.98±0.41 2.04±0.65 (5) LNP1023 G502 2.92±1.68 6.28±7.18 6.06±2.31 3.62±4.68 2.00±1.21 (6) LNP1024 G509 1.43±0.62 2.64±1.92 7.72±11.96 0.45±0.19 0.88±0.79 (7) LNP1024 G509 1.35±0.74 2.64±2.35 1.71±0.41 0.36±0.58 0.51±0.32 (8) LNP1025 G509 1.64 2.68 25.65 0.58 2.00 (9) LNP1021 G502 (10) LNP1022 0.56 6.15 28.80 0.85 0.61 G502 1.76 8.66 2907.86 11.26 1.72

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Table 24

MCP-1 measurements from Study 2

Treatment group Preliminary blood sampling 90 min 6 hours 24 hours 7 days (1)TSS 204.01±46, 39 197.62±19.5 4 310.84±45.87 179.07±20.77 234.61±71.79 (2) LNP1021 G502 303.67±36, 37 337.63±195, 18 755.20±581.4 5 339.75±206.2 0 214.82±40.81 (3) LNP1021 G502 229.30 358.10 3182.00 413.56 178.30 (4) LNP1022 G502 393.63±187.81 467.72±221, 61 1852.94±219 9.66 497.12±412.3 0 382.19±67.27 (5) LNP1023 G502 213.72±8.8 5 196.18±62.8 1 1722.18±141 3.90 197.83±74.01 156.16±18.87 (6) LNP1024 G509 237.76±96, 36 210.37±95.1 7 468.53±250.4 2 22.32±69.06 141.20±71.90 (7) LNP1024 G509 207.36 183.07 1885.66 235.70 163.11 (8) LNP1025 G509 259.57±112.98 299.21±304, 89 1193.10±974, 04 258.82±88.53 219.86±219.86 (9) LNP1021 G502 199.29 286.04 2001.23 197.57 196.44 (YU) LNP1022 G502 305.81 970.65 7039.06 8379.05 203.47

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Table 25

Complement C3a measurements from Study 3

Treatment group Preliminary blood sampling 90 min 6 hours 24 hours 7 days (1)TSS 42.47±10.30 55.40±13.58 29.30±14.46 41.70±23.65 27.43±12.43 (2) LNP10 21 G502 34.37±0.50 86.50±3.66 90.07±4.85 56.60±2.25 32.53±0.93 (3) LNP10 21 G502 34.30 128.00 93.30 33.40 28.20 (4) LNP10 22 G502 41.55±13.51 151.37±109.98 82.00±31.82 45.57±18.58 32.77±6.45 (5) LNP10 23 G502 31.67±3.19 74.40±22.08 74.13±48.61 33.83±9.75 27.70±8.05 (6) LNP10 24 G509 56.60±25.61 100.37±77.95 74.73±70.15 55.20±48.34 49.97±39.94 (7) LNP10 24 G509 33.80 33.90 33.70 26.10 20.90 (8) LNP10 25 G509 39.90±13.01 75.73±1.38 46.13±30.56 25.00±3.80 23.90±7.18 (9) LNP10 21 G502 34 85.70 133.00 62.00 25.50 (YU) LNP10 22 G502 29.8 68.10 113.00 71.70 23.30

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Table 26

bb complement measurements from study 3

Treatment group Preliminary blood sampling 90 min 6 hours 24 hours 7 days (1)TSS 1.46±0.70 2.18±0.78 1.96±0.64 0.945±0.1 5 1.34±0.50 (2) LNP1021 G502 1.77±0.60 6.51±3.66 11.00±4.8 5 3.59±2.25 2.07±0.93 (3) LNP1021 G502 1.24 2.90 11.50 2.97 1.24 (4) LNP1022 G502 1.52±0.34 5.67±2.28 10.2±3.36 3.66±1.68 1.84±0.24 (5) LNP1023 G502 1.65±0.94 4.4±1 7.68±4.67 2.64±1.18 2.08±1.32 (6) LNP1024 G509 1.61=1=0.13 4.52±1.81 4.50±3.22 1.63±0.84 1.63±0.32 (7) LNP1024 G509 0.96 2.99 2.64 1.13 1.07 (8) LNP1025 G509 1.37±0.17 4.9±4.51 3.79±3.84 1.66±1.43 1.35±0.44 (9) LNP1021 G502 1.41 5.67 11.50 4.64 1.38 (10) LNP1022 G502 1.28 5.22 14.10 5.64 1.87

Example 6: Screening of PEG lipids.

Another study compared alternative PEG lipids in LNP formulations containing 2 mol% or 3 mol% PEG lipid.

Three PEG lipids were used in the study: Lipid 1 (DMG-PEG2k; Nof) is depicted as:

ABOUT

DMG-PEG

Lipid 2, synthesized as described in Heyes, et al., J. Controlled Release 107 (2005), pp. 278-279 (see Synthesis of PEG2000-C-DMA), can be depicted as:

PEG-C-DMA and Lipid 3, disclosed in WO 2016/010840 (see connection S027, paragraphs [00240] - [00244]) and WO 2011/076807, can be depicted as:

..... .. .. .... ...... ..... ...... -... ...... I iC I .- ·... - ..· ...... ·....- 'y y y -. - ] _{| CH}

Lipid A was formulated with each PEG lipid at 2 mol% and 3 mol%. The lipid nanoparticle components were dissolved in 100% ethanol with the lipid component molar ratios given above. Briefly, RNA cargo was prepared in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in an RNA cargo concentration of approximately 0.45 mg/ml. LNPs were formulated with an aminolipid to RNA phosphate (N:P) molar ratio of about 4.5 with an mRNA to gRNA mass ratio of 1:1.

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Table 27

LNP No. LNP 784 LNP 785 LNP 786 LNP 787 LNP 788 LNP 789 CL/cholesterol/DSF X/PEG (theoretical mol.%) 45/44/9/2 45/43/9/3 45/44/9/2 45/43/9/3 45/44/9/2 45/43/9/3 mRNA Cas9 SEQ ID NO:48 Cas9 SEQ ID NO:48 Cas9 SEQ ID NO:48 Cas9 SEQ ID NO:48 Cas9 SEQ ID NO:48 Cas9 SEQ ID NO:48 gRNA G282 G282 G282 G282, G282, G282 SEQ ID NO:42 SEQ ID NO:42 SEQ ID NO:42 SEQ ID NO:42 SEQ ID NO:42 SEQ ID NO:42 PEG type Lipid 1 Lipid 1 Lipid 2 Lipid 2 Lipid 3 Lipid 3 N/P 4.5 4.5 4.5 4.5 4.5 4.5

Cas9, sg282 and LNP mRNA were prepared as described in example 1.

LNP compositions with Lipid 1, Lipid 2, or Lipid 3 were administered to female CD-1 mice and evaluated as described in Example 1 at doses of 1 mg/kg and 0.5 mg/kg body weight. Cohorts of mice were measured for liver editing by next generation sequencing (NGS) and serum TTR levels according to the methods of Example 1.

In fig. 6A and 6B compare serum TTR levels between PEG lipid formulations. In fig. 6A shows serum TTR in μg/ml, and FIG. 6B shows the data as percentage knockdown (%TSS). In fig. Figure 6C shows the percentage of editing achieved in the liver. The data indicate that LNP formulations with each of the PEG lipids tested were tested for effectiveness at 2 mol% and 3 mol%, with Lipid 1 consistently performing slightly better than Lipid 2 and Lipid 3.

Example 7. Analogs of Lipid A.

A number of structural analogues of Lipid A have been synthesized and tested in the LNP compositions described herein.

Synthesis: Lipid A is prepared by reacting 4,4-bis(octyloxy)butanoic acid (intermediate 13b in example 13 of WO 2015/095340) with (9Z, 12Z)-3-hydroxy-2(hydroxymethyl)propylooctadeca-9,12-dienoate (intermediate 13c) before adding the end group by reacting the product of intermediate 13b and intermediate 13c with 3-diethylamino-1-propanol. (See pp. 84-86 WO 2015/095340).

Intermediate 13b from WO 2015/095340 (4,4-bis(octyloxy)butanoic acid) was synthesized via 4,4-bis(octyloxy)butanenitrile as follows:

Intermediate 13a: 4,4-bis(octyloxy)butanenitrile

Pyridinium ptoluenesulfonate (748 mg, 3.0 mmol) was added to a mixture of 4,4-diethoxybutanenitrile (9.4 g, 60 mmol) and octan-1-ol (23.1 g, 178 mmol) at room temperature. The mixture was heated to 105°C and stirred for 18 hours in a reaction vessel open to air and not equipped with a reflux condenser. The reaction mixture was then cooled to room temperature and purified on silica gel (0-5% ethyl acetate in hexane gradient) to give 10.1 g (31.0 mmol) of intermediate 13a as a clear oil. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.55 (t, J=5.3 Hz, 1H), 3.60 (dt, J=9.2, 6.6 Hz, 2H), 3, 43 (dt, J=9.2, 6.6 Hz, 2H), 2.42 (t, J=7.4 Hz, 2H), 1.94 (td, J=7.4, 5.3 Hz , 2H), 1.63-1.50 (m, 4H), 1.38-1.19 (m, 20H), 0.93-0.82 (m, 6H) ppm.

31 mL of aqueous potassium hydroxide (2.5 M, 30.9 mL, 77.3 mmol) was then added to a solution of intermediate 13a (8.42 g, 31 mmol) in ethanol (30 mL) at room temperature. After equipping the vessel with a reflux condenser, the mixture was heated to 110°C and stirred for 24 hours. The mixture was then cooled to room temperature, acidified with aqueous hydrochloric acid (1H) to pH 5 and extracted three times into hexanes. The combined organic extracts were washed with water (twice) and brine, dried over anhydrous magnesium sulfate and concentrated in vacuo to give 8.15 g (23.6 mmol) of intermediate 13b as a clear oil, which was used without further

- 54 047139 additional cleaning. ¹ H-NMR (400 MHz, CDC13) δ 4.50 (t, J=5.5 Hz, 1H), 3.57 (dt, J=9.4, 6.7 Hz, 2H), 3.41 (dt, J=9.3, 6.7 Hz, 2H), 2.40 (t, J=7.4 Hz, 2H), 1.92 (td, J=7.4, 5.3 Hz, 2H), 1.56 (m, 4H), 1.37-1.21 (m, 20H), 0.92-0.83 (m, 6H) ppm. (structure below).

Intermediate 13b

Using the methods described above, C(5, 6, 7, 9 and 10)acetalcarboxylic acid intermediates, referred to as intermediates B3-F3 and listed below, were prepared using the appropriate alkan-1-ol reagents.

Intermediate B3 4,4-bis(pentyloxy)butanoic acid

¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.52 (t, J=5.5 Hz, 1H), 3.58 (dt, J=9.3, 6.6 Hz, 2H), 3, 41 (dt, J=9.3, 6.7 Hz, 2H), 2.45 (t, J=7.4 Hz, 2H), 1.94 (m, 2H), 1.57 (m, 4H ), 1.32 (m, J=3.7 Hz, 8H), 0.95-0.83 (m, 6H) ppm.

Intermediate C3: 4,4-bis(hexyloxy)butanoic acid

¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.44 (t, J=5.6 Hz, 1H), 3.49 (dt, J=9.3, 6.9 Hz, 2H), 3, 39 (dt, J=9.3, 6.8 Hz, 2H), 2.12 (t, J=7.6 Hz, 2H), 1.79 (kv, J=7.0 Hz, 2H), 1, 54 (m, 4H), 1.29 (m, 12H), 0.94-0.82 (m, 6H) ppm.

Intermediate D3: 4,4-bis(heptyloxy)butanoic acid

1H-NMR (400 MHz, CDCl ₃ ) δ 8.85 (br.s, 1H), 4.46 (t, J=5.6 Hz, 1H), 3.52 (dt, J=9.4, 6.8 Hz, 2H), 3.39 (dt, J=9.3, 6.8 Hz, 2H), 2.26 (t, J=7.6 Hz, 2H), 1.85 (kv, J=7.0 Hz, 2H), 1.53 (m, 4H), 1.29 (m, 16H), 0.94-0.80 (m, 6H) ppm.

Intermediate E3: 4,4-bis(nonyloxy)butanoic acid

¹ H-NMR (400 MHz, CDC13) δ 5.32 (br.s, 1H), 4.44 (t, J=5.6 Hz, 1H), 3.49 (dt, J=9.3, 6.9 Hz, 2H), 3.38 (dt, J=9.4, 6.9 Hz, 2H), 2.10 (t, J=7.6 Hz, 2H), 1.78 (kv, J=7.0 Hz, 2H), 1.53 (m, 4H), 1.27 (m, 24H), 0.88 (t, J=6.6 Hz, 6H) ppm.

Intermediate F3: 4,4-bis(decyloxy)butanoic acid: ' O

OH ,0 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.48 (t, J=5.5 Hz, 1H), 3.55 (m, 2H), 3.42 (m, 2H), 2 .29 (dd, J=10.8, 7.5 Hz, 2H), 1.90-1.82 (m, 2H), 1.55 (m, 4H), 1.27 (m, 28H), 0.88 (t, J=6.7 Hz, 6H) ppm

Acetal analogues of Lipid A (C(8)) were synthesized by reacting intermediates C(5, 6, 7, 9 or 10)-acetalcarboxylic acid (B3-F3) with intermediate 13c before reacting the product of this step with 3-diethylamino- 1-propanol. (See pp. 84-86 WO2015/095340). Each analog was synthesized and characterized by ¹ H-NMR (data not shown).

Analogues C7, C9 and C10 were composed of 45 mol% Lipid A analogue, 2 mol% DMG-PEG2k, 9 mol% DSPC and 44 mol% cholesterol with an N:P ratio of 4.5. Each analogue was also composed of 55 mol% Lipid A analogue, 2.5 mol% DMG-PEG2k, 9 mol% DSPC and 38.5 mol% cholesterol with an N:P ratio of 6. The lipid nanoparticle components were dissolved in 100% ethanol with the molar ratios of the lipid component given above. RNA cargo was prepared in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in an RNA cargo concentration of approximately 0.45 mg/ml.

The RNA cargo contains Cas9 mRNA containing SEQ ID NO: 43 and sg282 prepared as described above. LNPs were formulated as described in Example 1.

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An expanded panel of acetal analogues, including LNP compositions containing C(5) and C(6) Lipid A analogues, were tested in conjunction with the previous panel. The two new analogs were formulated with 55 mol% Lipid A analogue, 2.5 mol% DMG-PEG2k, 9 mol% DSPC and 33.5 mol% cholesterol with an N/P ratio of 6 as described above. The analysis demonstrated that all LNPs were smaller than 120 nm, PDI was lower than 0.2, and % encapsulated RNA was higher than 80%. The results of analyzes for the compositions are presented in table. 28 below.

Table 28

IN LNP Analogs Lipid A Analogue, mol % PEG % N/P RNA Conc, (mg/m l) %HER Z- avg. (nm) PDI Average value (nm) LNP 1122 Analog C 5 (LP000030001) 55 2.5 6 0.063 88 118.8 0.103 88.17 LNP 1123 Analogue C6 (LP000031001) 55 2.5 6 0.067 95 107.6 0.038 88.1 LNP 1004 Analog C7 (LP000020001) 55 2.5 6 0.068 98 100 0.012 81.55 LNP 1002 LP000001- 011 55 2.5 6 0.067 98 95.06 0.01 78.95 LNP 1006 Analog C9 (LP000021001) 55 2.5 6 0.067 97 95.43 0.022 80.35 LNP 1008 Analogue SYU (LP000022001) 55 2.5 6 0.069 95 103.9 0.008 86.79

Analogues were assessed for pKa using 6-(p-toluidino)-6-naphthalenesulfonic acid (TNS) dissolved in water. In this assay, 0.1 M phosphate buffer was prepared at various pH values ranging from 4.5 to 10.5. Each analog was separately prepared in 100% ethanol. Lipid and TNS were then added to a separate pH buffer and transferred to the plate for analysis on a SpectraMax plate reader at a wavelength of 321-488 nm. The values were plotted to obtain pKa, logIC ₅₀ is used as pKa.

Female CD-1 mice were dosed as described in Example 1, 0.3 mg/kg (FIGS. 7A-7E) or 0.1 mg/kg (FIGS. 7F-7G). Briefly, female CD-1 mice from Charles River Laboratories, n=5 per group, were administered LNP compositions at various doses. At necropsy (7 days post-dose), serum was collected for TTR analysis and liver was collected for editing analysis. Serum TTR analysis and percent editing were performed as described in Example 1. Serum TTR levels and liver editing from FIG. 7A-7E indicate that all analogues acted comparable to Lipid A at 0.3 milligrams per kilogram of body weight. In fig. 7B-fig. 7G shows that although Lipid A had the greatest potency, all newly synthesized analogues have suitable TTR knockdown and liver editing.

Example 8: Dose Response Curve - Cynomolgus Macaque Primary Hepatocytes

Primary liver hepatocytes. Primary cynomolgus liver hepatocytes (CLM) (Gibco) were thawed and resuspended in hepatocyte thawing medium with supplements (Gibco, cat. CM7000) followed by centrifugation at 80 g for 4 minutes. The supernatant was discarded, and the pelleted cells were resuspended in hepatocyte growth medium with a complex of additives (Invitrogen, cat. A1217601 and CM3000). Cells were counted and seeded onto BioCoat Collagen I 96-well plates (ThermoFisher, cat. 877272) at a density of 50,000 cells/well. The seeded cells were allowed to settle and adhere for 24 hours in a tissue culture incubator (37°C and 5% CO ₂ atmosphere) before applying LNP. After incubation, the cells were checked for monolayer formation and the medium was replaced with hepatocyte culture medium with serum-free supplement complex (Invitrogen, cat. A1217601 and CM4000).

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The LNP formulations for this study (LNP1021, LNP1022, LNP1023, LNP1024, LNP1025, and LNP897) were prepared as described above.

Various doses of lipid nanoparticle formulations containing modified ssRNAs were tested on primary cynomolgus monkey hepatocytes to obtain a dose response curve. After seeding and 24-hour culture, LNPs were incubated in hepatocyte maintenance medium containing 6% cynomolgus serum at 37°C for 5 minutes. After incubation, LNP was added to primary cynomolgus macaque hepatocytes in an 8-point 2× dose response curve, starting with 100 ng of mRNA. Cells were lysed 72 hours after treatment for NGS analysis as described in Example 1. Percentage editing was determined for various LNP compositions and the data are presented in FIG. 8A. The % editing value by Cas9 mRNA (SEQ ID NO: 48) and U-depleted Cas9 mRNA (SEQ I NO: 43) is presented in FIG. 8B. The LNP compositions are described in table. 2 (LNP 897) and table. 5 (LNP 1021, 1022, 1023, 1024 and 1025).

The results demonstrate quantitative analysis results for comparative efficacy assessments, which demonstrate that both mRNA and LNP composition influence efficacy.

Example 9. RNA cargo: joint compositions of mRNA and gRNA.

This study assessed the in vivo efficacy of different ratios of gRNA to mRNA in mice. CleanCap™ capped Cas9 mRNAs with ORF SEQ ID NO: 4, 5'-UTR HSD, human albumin 3'-UTR, Kozak sequence and poly-A tail were produced by IVT synthesis as described in Example 1 with triphosphate M -methylpseudouridine instead of uridine triphosphate.

LNP formulations were prepared from the described mRNA and sg282 (SEQ ID NO: 42; G282) as described in example 2, with Lipid A, cholesterol, DSPC and PEG2k-DMG in a molar ratio of 50:38:9:3 and N:P ratio , equal to 6. The mass ratios of Cas9 mRNA:gRNA in the compositions were as shown in Table. 29.

Table 29

ID LNP Ratio Guide: Cas9 mRNA (w/w) Conc. RNA (mg/ml) HER (%) Z-average size (nm) PDI particles Number average (nm) 1110 8:1 0.92 99 69.52 0.022 56.47 1111 4:1 0.86 97 76.65 0.065 57.36 1112 2:1 0.90 99 76.58 0.036 63.11 1113 1:1 0.97 99 76.60 0.071 58.92 1114 1:2 1.05 99 76.34 0.018 62.82 1115 1:4 0.65 99 82.64 0.018 66.63 1116 1:8 0.75 100 82.01 0.039 65.05

For in vivo characterization, the above LNPs were administered to mice at 0.1 mg total RNA (mg guide RNA+mg mRNA) per kg (n=5 per group). 7-9 days after dosing, animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured as described in Example 1. Serum TTR and liver editing results are shown in FIG. 9A and 9B. Negative control mice were injected with TSS vehicle.

In addition, the above LNPs were administered to mice at a constant mRNA dose of 0.05 mg mRNA per kg (n=5 per group), while varying the mRNA dose from 0.06 mg per kg to 0.4 mg per kg. 7-9 days after dosing, animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured. Serum TTR and liver editing results are shown in Fig. 9C and 9D. Negative control mice were injected with TSS vehicle.

Example 10. Neutral lipids.

To evaluate the in vivo efficacy of LNPs, LNP formulations with the mRNA from Example 2 and sg534 (SEQ ID NO: 72; G534) were prepared as described in Example 2. The lipid nanoparticle components were dissolved in 100% ethanol at the lipid component molar ratios given below. Briefly, RNA cargo was prepared in a buffer of 25 mM citrate and 100 mM NaCl at pH 5.0, resulting in an RNA cargo concentration of approximately 0.45 mg/ml. LNPs were formulated with an aminolipid to RNA phosphate (N:P) molar ratio of about 6 with a gRNA to mRNA mass ratio of 1:2.

LNP formulations were analyzed for average particle size, polydispersity (pdi), total

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RNA and RNA encapsulation efficiency as described in Example 1. Analysis of average particle size, polydispersity (PDI), total RNA content and RNA encapsulation efficiency are shown in Table. 30. Lipid molar ratios are presented as aminolipid (Lipid A)/neutral lipid/auxiliary lipid (cholesterol)/PEG-lipid (PEG2k-DMG). Neutral lipid was DSP, DPPC, or none as indicated.

Table 30

LNP compositions and their data. (Molar ratios of lipids are presented as aminolipid (Lipid A)/neutral lipid/auxiliary lipid (cholesterol)/PEG-lipid (PEG2k-DMG))

IN sample Nate ral lipid Molar ratios RNA END (mg/m l) %HER Z- avg. (nm) PDI Mean sign (nm) % edited in the liver Serum TTP (µg/ml) % nokd auna TTR Control TSS 0.0 1248.9 CO241 - 50.0/0.0/ 47.0/3.0 1.46 94 75.64 0.090 54.21 1.8 1070.2 14.3 CO242 - 59.0/0.0/ 38.0/3.0 1.51 94 92.25 0.019 75.56 12.0 819.6 34.4 CO243 - 54.5/0.0/ 42.5/3.0 1.62 94 78.90 0.052 61.49 3.3 1260.5 -0.9 CO244 DSF X 50.0/9.0/ 39.0/2.0 1.50 93 101.3 0.044 80.73 27.4 741.0 40.7 CO034 DSFH 50.0/9.0/ 38.0/3.0 1.48 97 84.23 0.040 66.96 34.2 630.1 49.6 CO245 DSF X 52.5/4.0/ 42.5/3.0 1.55 95 81.88 0.054 64.54 5.8 846.6 32.2 CO246 DFT X 50.0/9.0/ 38.0/3.0 1.52 96 87.11 0.040 70.04 35.9 528.6 57.7 CO247 DFT X 52.5/4.0/ 42.5/3.0 1.54 97 83.67 0.050 66.43 18.3 726.6 41.8

For in vivo characterization, the above LNPs were administered intravenously to female SpregDawley rats in an amount of 0.3 mg of total RNA (guide RNA and mRNA) per kg body weight. There were five rats in the group. Seven days after dosing, animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured as described in Example 1. Negative control animals were dosed with TSS vehicle. Serum TTR and liver editing results are shown in Fig. 10A and 10B, and in table. 30 (above).

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Brief description of the disclosed sequences

SEQ ID NO Description 1 Cas9 DNA coding sequence using a thymidine analogue for the minimum uridine content codons listed in Table 3, with start and stop codons 2 DNA coding sequence of Cas9 using codons with typically high expression in humans 3 Amino acid sequence of Cas9 with one nuclear localization signal (IxNLS) as the C-terminal 7 amino acids 4 Cas9 mRNA ORF using minimum uridine content codons as listed in Table 3, with start and stop codons 5 Cas9 mRNA ORF using codons typically highly expressed in humans, with start and stop codons 6 Amino acid sequence of Cas9 nickase with IxNLS as C-terminal 7 amino acids 7 Cas9 nickase mRNA ORF encoding SEQ Ш NO: 6, using codons with minimal uridine content, as indicated in Table 3, with start and stop codons 8 Amino acid sequence of dCas9 with IxNLS as the Terminal 7 amino acids 9 dCas9 mRNA ORF encoding SEQ Ш NO: 8, using

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codons with minimal uridine content, as indicated in the table 3, with start and stop codons 10 Cas9 mRNA coding sequence using the minimum uridine content codons listed in Table 3 (no start or stop codons; suitable for inclusion in a fusion protein coding sequence) eleven Cas9 nickase mRNA coding sequence using the minimum uridine content codons listed in Table 3 (no start or stop codons; suitable for inclusion in a fusion protein coding sequence) 12 dCas9 mRNA coding sequence using the minimum uridine content codons listed in Table 3 (no start or stop codons; suitable for inclusion in a fusion protein coding sequence) 13 Amino acid sequence of Cas9 (no NLS) 14 Cas9 mRNA ORF encoding SEQ Ш NO: 13, using codons with minimal uridine content, as indicated in Table 3, with start and stop codons 15 Ca89 coding sequence encoding SEQ III NO: 13, using the minimum uridine content codons listed in Table 3 (no start or stop codons; suitable for inclusion in the fusion protein coding sequence) 16 Amino acid sequence of Cas9 nickase (without NLS) 17 Cas9 nickase mRNA ORF encoding SEQ GO NO: 16, using codons with minimal uridine content, as indicated in Table 3, with start and stop codons 18 Cas9 nickase coding sequence coding for SEQ GO NO: 16, using the minimum uridine content codons listed in Table 3 (no start or stop codons; suitable for inclusion in a fusion protein coding sequence)

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19 Amino acid sequence of dCas9 (without NLS) 20 dCas9 mRNA ORF encoding SEQ Ш NO: 13, using codons with a minimum uridine content, as indicated in Table 3, with start and stop codons 21 dCas9 coding sequence encoding SEQ III NO: 13, using the minimum uridine content codons listed in Table 3 (no start or stop codons; suitable for inclusion in the fusion protein coding sequence) 22 Amino acid sequence of Cas9 with two nuclear localization signals (2xNLS) as C-terminal amino acids 23 Cas9 mRNA ORF encoding SEQ Ш NO: 13, using codons with minimal uridine content, as indicated in Table 3, with start and stop codons 24 Ca89 coding sequence encoding SEQ III NO: 13, using the minimum uridine content codons listed in Table 3 (no start or stop codons; suitable for inclusion in the fusion protein coding sequence) 25 Amino acid sequence of Cas9 nickase with two nuclear localization signals as C-terminal amino acids 26 Cas9 nickase mRNA ORF encoding SEQ GO NO: 16, using codons with minimal uridine content, as indicated in Table 3, with start and stop codons 27 Cas9 nickase coding sequence coding for SEQ GO NO: 16, using the minimum uridine content codons listed in Table 3 (no start or stop codons; suitable for inclusion in a fusion protein coding sequence) 28 Amino acid sequence of dCas9 with two nuclear localization signals as C-terminal amino acids 29 dCas9 mRNA ORF encoding SEQ GO NO: 13 using codons with minimal uridine content as indicated in the table

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3, with start and stop codons thirty yCa89 coding sequence encoding SEQ III NO: 13, using the minimum uridine content codons listed in Table 3 (no start or stop codons; suitable for inclusion in the fusion protein coding sequence) 31 T7 promoter 32 5'-UTR of human beta-globin 33 Z'-UTR of human beta-globin 34 5'-UTR of human alpha globin 35 Z'-UTR of human alpha globin 36 Xenopus laevis beta-globin 5'-UTR 37 Z'-UTR of beta-globin Xenopus laevis 38 5'-UTR of bovine growth hormone 39 Z'-NTO of bovine growth hormone 40 Z'-UTR alpha, mature chain 1 of hemoglobin Mus musculus (Hba-al) 41 5'-UTR HSD17B4 42 Single guide RNA G282 targeting the mouse TTR gene 43 Cas9 transcript with 5'-UTR HSD, ORF corresponding to SEQ III NO: 4, Kozak sequence and Z’-NTO ALB 44 Cas9 transcript with 5'-UTR HSD, ORF corresponding to SEQ III NO: 4, and Z’-NTO ALB 45 Alternative Cas9 ORF with 19.36% U content 46 Cas9 transcript with 5'-UTR HSD, ORF corresponding to SEQ Ш NO: 45, Kozak sequence and 3'-UTR ALB 47 Cas9 transcript with 5'-UTR HSD, ORF corresponding to SEQ III NO: 45, and 3'-UTR ALB 48 Cas9 transcript containing a Cas9 ORF using codons typically highly expressed in humans

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49 Cas9 transcript containing a Kozak sequence with a Cas9 ORF using codons typically highly expressed in humans 50 Cas9 ORFs with deleted splice points; U content 12.75% 51 Cas9 transcript with 5'-UTR HSD, ORF corresponding to SEQ Ш NO: 50, Kozak sequence and 3'-UTR ALB 52 Cas9 ORFs with minimal uridine content codons frequently used in humans in general; U content 12.75% 53 Cas9 transcript with 5'-UTR HSD, ORF corresponding to SEQ Ш NO: 52, Kozak sequence and 3'-UTR ALB 54 Cas9 ORFs with minimal uridine content codons rarely used in humans in general; U content 12.75% 55 Cas9 transcript with 5'-UTR HSD, ORF corresponding to SEQ Ш NO: 54, Kozak sequence and 3'-UTR ALB 56 Cas9 transcript with AGG as the first three nucleotides for use with CleanCap™, 5'-UTR of HSD, ORF corresponding to SEQ III NO: 4, Kozak sequence and 3'-UTR of ALB 57 Cas9 transcript with 5'-UTR from CMV, ORF corresponding to SEQ ID NO: 4, Kozak sequence and 3'-UTR of ALB 58 Cas9 transcript with 5'-UTR from HBV, ORF corresponding to SEQ ID NO: 4, Kozak sequence and 3'-UTR of HBV 59 Cas9 transcript with 5'-UTR from XBG, ORF corresponding to SEQ ID NO: 4, Kozak sequence and 3'-UTR of XBG 60 Cas9 transcript with AGG as the first three nucleotides for use with CleanCap™, 5'-UTR of XBG, ORF corresponding to SEQ III NO: 4, Kozak sequence and 3'-UTR of XBG 61 Cas9 transcript with AGG as the first three nucleotides for use with CleanCap™, 5'-UTR from HSD, ORF corresponding to SEQ III NO: 4, Kozak sequence and 3'-UTR of ALB 62 30/30/39 poly-A sequence 63 poly-A sequence 100

- 63 047139

64 Single guide RNA G209 targeting the mouse TTR gene 65 ORF encoding Neisseria meningitidis Cas9 using minimal uridine content codons as listed in Table 3, with start and stop codons 66 An ORF encoding Neisseria meningitidis Cas9 using the minimum uridine content codons listed in Table 3 (no start or stop codons; suitable for inclusion in a fusion protein coding sequence) 67 Transcript containing SEQ GO NO: 65 (encoding Neisseria meningitidis Cas9) 68 Amino acid sequence of Neisseria meningitidis Cas9 69 Single guide RNA G390 targeting the rat TTR gene 70 Single guide RNA G502 targeting the cynomolgus TTR gene 71 Single guide RNA G509 targeting the cynomolgus TTR gene 72 Single guide RNA G534 targeting the rat TTR gene

See the sequence table below for the sequences themselves. Transcript sequences typically contain GGG as the first three nucleotides for use with ARCA or AGG as the first three nucleotides for use with CleanCap™. Accordingly, the first three nucleotides can be modified for use with other capping approaches, such as the vaccinia virus capping enzyme. Promoters and poly-A sequences are not included in the transcript sequence. A promoter, such as the T7 promoter (SEQ ID NO: 31), and a poly-A sequence, such as SEQ ID NO: 62 or 63, can be attached to the described transcript sequences at the 5' and 3' ends, respectively. Most nucleotide sequences are in the form of DNA, but can be easily converted to RNA by changing T to U.

Sequence table.

The following sequence table provides a list of the sequences described in this document. It is clear that if a DNA sequence (containing a T) is referred to in relation to RNA, then the T should be replaced by a U (which may or may not be modified depending on the context), and vice versa.

- 64 047139

Description Subsequence SEQ ID No. Cas9 DNA coding sequence 2 ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAAC AAACAGCGTCGGATGGGCAGTCATCACAGACGAATACA AGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACA GACAGACACAGCATCAAGAAGAACCTGATCGGAGCACT GCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGAC TGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAA GAACAGAATCT GCTACCTGCAGGAAATCTTCAGCAACG AAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTG GAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGA AAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCG CATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAG ACTGATCTACCTGGCACTGGCACACAT ATC GCACTGAGCCTGGGACTGACACCGAACTTCAAGAG CAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGA GCAAGGACACATACGACGACGACCTGGACAACCTGCTG GCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGC AGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACA TCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTG AGCGCAAGCATGATCAAGAG ATACGACGAACACCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGC TGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGC AAGAACGGATACGCAGGATACATCGACGGAGGAGCAAG CCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGA AAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGA ACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTC GAC 1

- 65 047139

AACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACT GCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTT CCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGA CATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAG GAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGA CAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGA CAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTG CCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAAT GAGAAAGCCGGCATTCCTGAGCGGAACAGAAGAAGG CAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTC ACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGA AGAT CGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAG ACAGATTCAACGCAAGCCTGGGAACATACCACGACCTG CTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAACGAAGACATCCTGGAAGACATCGTCCTGACAC TGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATG GCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAG CAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGA CGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGA CGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCAC AGGTCAGCGGACAGGGACAGCCTGCACGAACACATC GCAAACCTGGCAGGAAGCCCG GCAATCAAGAAGGGAAT CCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGG TCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAA ATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGA AGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGG AATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACC CGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTAC CTG TACTACCTGCAGAACGGAAGAGACATGTACGTCGA CCAGGAACTGGACATCAACAGACTGAGCGACTACGACG TCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACA

- 66 047139

GCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAAC AGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGT CAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACG CAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACA AAGGCAGAGAGGAGGACTGAGCGAACTGGACAAGG CAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAG ATCACAAAGCACG TCGCACAGATCCTGGACAGCAGAAT GAACACAAAGTACGACGAAAACGACAAGCTGATCAGAG AAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGC GACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGA AATCAACAACTACCACCACGCACACGACGCATACCTGA ACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCG AAGCTGGAAAGCGAATTCGTCTACGGA GACTACAAGGT CTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGG AAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGC AACATCATGAACTTCTTCAAGACAGAAATCACACTGGCA AACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAA CGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGA GACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCA GGTCAA CATCGTCAAGAAGACAGAAGTCCAGACAGGAG GATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGC GACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACA GCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGC AAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCAC AATCATGGAAAGAAGCAGCTTCG AAAAGAACCCGATCG ACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAG GACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAA CTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCA AGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAA AGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAG CA GCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAAT CATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCC TGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATAC

- 67 047139

AACAAGCACAGACAAGCCGATCAGAGAACAGGCAG AAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCA ACACTGATCCACCAGAGCATCACAGGACTGTACGAAAC AAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAG GAAGCCCGAAGAA GAAGAGAAAGGTCTAG Cas9 DNA coding sequence 1 ATGGATAAGAAGTACTCAATCGGGCTGGATATCGGAAC TAATTCCGTGGGTTGGGCAGTGATCACGGATGAATACAA AGTGCCGTCCAAGAAGTTCAAGGTCCTGGGGAACACG ATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCTG CTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTC AAACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAA TCGCATCT GCTATCTGCAAGAGATCTTTTCGAACGAAAT GGCAAAGGTCGACGACAGCTTCTTCCACCGCCTGGAAG AATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGG CATCCTATCTTTGGAAACATCGTCGACGAAGTGGCGTAC CACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAA GTTGGTTGACTCAACTGACAAGGCCGACCTCAGATTGAT CTACTTGGCCCTCGCCCATAT GATCAAATTCCGCGGACA CTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGA CGTGGATAAGCTTTTCATTCAACTGGTGCAGACCTACAA CCAACTGTTCGAAGAAAACCCAATCAATGCTAGCGGCGT CGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTC GCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAG AGAAAAAGAACGGACTTTTCGGCAACTTGATCG CTCTCT CACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACC TGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACC TACGACGACGACTTGGACAATTTGCTGGCACAAATTGGC GATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTT TCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAAC ACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATT AAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTC AAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAA GGAGATCTTCTTCGACCAGTCCAAGAATGGGTACGCAGG 2

- 68 047139

GTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATA AGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACC GAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCT CCGGAAACAGAGAACCTTTGACAACGGATCCATTCCCCA CCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCG CCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGA AAAGATCGAGAAAA TTCTGACGTTCCGCATCCCGTATTA CGTGGGCCCACTGGCGCGCGGCAATTCGCCTTCCGTG GATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGA ATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAA AGCTTCATCGAACGAATGACCAACTTCGACAAGAATCTC CCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTAC GAATACTTCACTGTCTACAACGAACT GACTAAAGTGAAA TACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCC GGGAACAGAAGAAAGCAATTGTCGATCTGCTGTTCAA GACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGG ACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAA TCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGA ACCTATCATGATCTCCTGAAGATCATCAAGGACA AGGAC TTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGA TATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGAT GATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGA CGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACA CTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTA TTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCC TCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAAT TGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCC AAAAAGCACAAGTGTCCGGACAGGGAGACTCACTCCAT GAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAG AAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGCT GGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCG TGGCCCGAGAAAACCAGACTACCCCAGAAG GGCCAGAAAAACTCCCGCGAAAGGATGAAGCGGATCGA AGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAG AGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAG

- 69 047139

CTCTACCTGTACTATTTGCAAAATGGACGGGACATGTAC GTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTAC GACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGAT GACTCGATCGATAACAAGGTGTTGACTCGCAGCGACAA GAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGG TCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTG GAAGCTGATTACCCAGAGAAAGTTTGACAATCTC ACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAA GGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGC AGATTACCAAGCACGTGGCGCAGATCTTGGACTCCCGCA TGAACACTAAATACGACGAGAACGATAAGCTCATCCGG GAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTC GGACTTTCGGAAGGACTTTCAG TTTTACAAAGTGAGAGA AATCAACAACTACCATCACGCGCATGACGCATACCTCAA CGCTGTGGTCGGTACCGCCCTGATCAAAAAGTACCCTAA ACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTA CGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAA TCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACA TCATGAACTTTTTCAAGACTGAAATTACGCTGGCCAAT g GATTCGCCGACTGTCGCATACTCCGTCCTC GTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAGCT CAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGA ACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGA GGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCA TCAAACTCCCCAAGTACTCACTGTTCGAACTGGAAAATG GTCGGAAGCGCATGCTGGC TTCGGCCGGAGAACTCCAA AAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAA CTTCCTCTATCTTGCTTCGCACTACGAAAAACTCAAAGG GTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGG

- 70 047139

AGCAGCACAAGCATTATCTGGATGAAATCATCGAACAA ATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCC AACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGA GATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCA CTTGTTCACCCTGACTAACCTGGGAGCCCCAGCCGCCTT CAAGTACTTCGATACTACTATCGATCGCAAAAGATACAC GTCCACCA AGGAAGTTCTGGACGCGACCCTGATCCACCA AAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTC GCAGCTGGGTGGCGATGGCGGTGGATCTCCGAAAAAGA AGAGAAAGGTGTAATGA Amino acid sequence of Cas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ LFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ EEFYKFIKPILEKMDGTEELLV KLNREDLLRKQRTFDNGSIP HQIHLGELHAILRRQEDFYPFLKDNREKIEKILFTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQK N SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSK SDFRKDFQFYKVREI NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD 3

- 71 047139

VRKMIAKSEQEIGKATAKYFFYSNIMNFKTEITLANGEIRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGDGGGSPKKKRKV Cas9 mRNA open reading frame (ORF) 2 AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAA CAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUA CAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAAC ACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAG CACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAAC AAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGA AGAAAGAACAGA AUCUGCUACCUGCAGGAAAUCUUCA GCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAG AAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCG ACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGG CAGACCUGAGACUGAUCUACCUGGCACU GGCACACAUG AUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACC UGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUC CAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAA ACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUC CUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAA ACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGG ACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUG ACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAG ACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGA CGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGU ACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGAC GCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAG AAAUCACAAAGGCACCGCU GAGCGCAAGCAUGAUCAA 4

- 72 047139

GAGAUACGACGAACACCACCAGGACCUGACACUGCUGA AGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAA GGAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCA GGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCU ACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGG AACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGA GAAAGCAGAGAACAUUCGACAACGGAAGCA UCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUC CUGAGAAGACAGGAACUUCUACCCGUUCCUGAAGG ACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAA CAAUCACACCGUGGAACUUCGA AGAAGUCGUCGACAA GGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCC GAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUAC AACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAA CAGAAAG GUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGA AGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGU CGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAAGGACAAGGACUUCCUGG ACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGU CCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUC CUGAAGACAUACGCACACCUGUUCGACG ACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACAC AGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGA AUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACU UCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAU GCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAA GACAUCCAGAAGGCACAGGUCAGCG GACAGGGAGACA GCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCG UCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCC

- 73 047139

GGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAG ACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAA UGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGC UGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAA CGGAAGACAUGUACGUCGACCAGGAACUGGACAUC AACAGACUGAGCGA CUACGACGUCGACCACAUCGUCCC GCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAG GUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCG ACAACGUCCCGAGCGAAGAAGUCGAAGAAGAUGAA GAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUC ACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGA GAGGAGGACUGAGCGAACUGGACAACA GGAUUCAU CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAG CACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAA AGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAA GGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUC AGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCA ACAACUACCACCACGCACACGACGCAUACCUGAACGCA GUCGU CGGAACAGCACUGAUCAAGAAGUACCCGAAGC UGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUA CGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAA AUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCA ACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGC AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACA AACGGAGAAACAGGAGAAAUC GUCUGGGACAAGGGAA GAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCC GCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACA GGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAA ACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGA CCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGGCAAAGGUCGAAAAGG GAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCU GGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGG

- 74 047139

AAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUA CAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUG CUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAAC UGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUG GCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAG ACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAA GCACUACCUG GACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGG ACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUG UUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAA GUACUUCGACACAACAAUCGACAGAAAGAGAUACACA AGCACAAAGGAAGUCCUGGACGCAA CACUGAUCCACCA GAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUG AGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGA AGAAGAGAAAGGUCUAG OPC 1 mRNA Cas9 AUGGAUAAGAAGUACUCAAUCGGGCUGGAUAUCGGAA CUAAUUCCGUGGGUUGGGCAGUGAUCACGGAUGAAUA CAAAGUGCCGUCCAAGAAGUUCAAGGUCCUGGGGAAC ACCGAUAGACACACAUCAAGAAAAAUCUCAUCGGAG CCCUGCUGUUUGACUCCGGCGAAACCGCAGAAGCGACC CGGCUCAAACGUACCGCGAGGCGACGCUACACCCGGCG GAAGAAUC GCAUCUGCUAUCUGCAAGAGAUCUUUUCG AACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACC GCCUGGAAGAAUCUUUCCUGGUGGAGGAGGACAAGAA GCAUGAACGGCAUCCUAUCUUUGGAAACAUCGUCGAC GAAGUGGCGUACCACGAAAAGUACCCGACCAUCUACCA UCUGCGGAAGAAGUUGGUUGACUCAACUGACAAGGCC AUUGAUCUACUUGGCCCUCGCCCAUAUGAU CAAAUUCCGCGGACACUUCCUGAUCGAAGGCGAUCUG AACCCUGAUAACUCCGACGUGGAUAAGCUUUUCAUUC AACUGGUGCAGACCUACAACCAACUGUUCGAAGAAAA CCCAAUCAAUGCUAGCGGCGUCGAUGCCAAGGCCAUCC UGUCCGCCCGGCUGUCGAAGUCGCGGCGCCUCGAAAAC CUGAUCGCACAG CUGCCGGGAGAGAAAAAGAACGGAC 5

- 75 047139

UUUUCGGCAACUUGAUCGCUCUCUCACUGGGACUCACU CCCAAUUUCAAGUCCAAUUUUGACCUGGCCGAGGACGC GAAGCUGCAACUCUCAAAGGACACCUACGACGACGACU UGGACAAUUUGCUGGCACAAUUGGCGAUCAGUACGC GGAUCUGUUCCUUGCCGCUAAGAACCUUUCGGACGCA AUCUUGCUGUCCGAUAUCCUGCGCGUGA ACACCGAAA UAACCAAAGCGCCGCUUAGCGCCUCGAUGAUUAAGCG GUACGACGAGCAUCACCAGGAUCUCACGCUGCUCAAAG CGCUCGUGACAGCAACUGCCUGAAAAGUACAAGGA GAUCUUCUUCGACCAGUCCAAGAAUGGGUACGCAGGG UACAUCGAUGGAGGCGCUAGCCAGGAAGAGUUCUAUA AGUUCAUCAAGCCAAUCCUGGAAAAGAUGGACGGAAC CUGCUGGUCAAGCUGAACAGGGAGGAUCUG CUCCGGAAACAGAGAACCUUUGACAACGGAUCCAUUCC CCACCAGAUCCAUCUGGGUGAGCUGCACGCCAUCUUGC GGCGCCAGGAGGACUUUUACCCAUUCCUCAAGGACAAC CGGGAAAAGAUCGAGAAAAUUCUGACGUUCCGCAUCC CGUAUUACGUGGGCCCACUGGCGCGCGGCAAUUCGCGC GUGGAUGACUAGAAAAUCAGAGGAAACCAUCA CUCCUUGGAAUUUCGAGGAAGUUGUGGAUAAGGGAGC UUCGGCACAAAGCUUCAUCGAACGAAUGACCAACUUC GACAAGAAUCUCCCAAACGAGAAGGUGCUUCCUAAGC ACAGCCUCCUUUACGAAUACUUCACUGUCUACAACGAA CUGACUAAAGUGAAAUACGUUACUGAAGGAAUGAGGA AGCCGGCCUUUCU GUCCGGAGAACAGAAGAAAGCAAU UGUCGAUCUGCUGUUCAAGACCAACCGCAAGGUGACC GUCAAGCAGCUUAAAGAGGACUACUUCAAGAAGAUCG AGUGUUUCGACUCAGUGGAAAUCAGCGGGGUGGAGGA CAGAUUCAACGCUUCGCUGGGAACCUAUUCAUGAUCUCC UGAAGAUCAUCAAGGACAAGGACUUCCUUGACAACGA GGAGAACGAGCAUCCUGGAAG AUAUCGUCCUGACC UUGACCCUUUUCGAGGAUCGCGAGAUGAUCGAGGAGA GGCUUAAGACCUACGCUCAUCUCUUCGACGAUAAGGU CAUGAAACAACUCAAGCGCCGCCGGUACACUGGUUGG GGCCGCCUCUCCCGCAAGCUGAUCAACGGUAUUCGCGA

- 76 047139

UAAACAGAGCGGUAAAACUAUCCUGGAUUUCCUCAAA UCGGAUGGCUUCGCUAAUCGUAACUUCAUGCAAUUGA UCCACGACGACAGCCUGACCUUUAAGGAGGACAUCCAA AAAGCACAAGUGUCCGGACAGGGACUCACUCCAUG AACACAUCGCGAAUCUGGCCGGUUCGCCGGCGAUUAA GAAGGGAAUUCUGCAAACUGUGAAGGUGGUCGACGAG CU GGUGAAGGUCAUGGGACGGCACAAACCGGAGAAUA UCGUGAUUGAAAUGGCCCGAGAAAACCAGACUACCCA GAAGGGCCAGAAAAACUCCCGCGAAAGGAUGAAGCGG AUCGAAGAAGGAAUCAAGGAGCUGGGCAGCCAGAUCC UGAAAGAGCACCCGGUGGAAAACACGCAGCUGCAGAA CGAGAAGCUCUACCUGUACUAUUUGCAAAAUGGACGG GACAUGUACGUGGAC CAAGAGCUGGACAUCAAUCGGU UGUCUGAUUACGACGUGGACCACAUCGUUCCACAGUCC UUUCUGAAGGAUGACUCGAUCGAUAACAAGGUGUUGA CUCGCAGCGACAAGAACAGAGGGAAGUCAGAUAAUGU GCCAUCGGAGGAGGUCGUGAAGAAGAUGAAGAAUUAC UGGCGGCAGCUCCUGAAUGCGAAGCUGAUUACCCAGA GAAAGUUUGACAAUCUCACUAAA GCCGAGCGCGGCGG ACUCUCAGAGCUGGAUAAGGCUGGAUUCAUCAAACGG CAGCUGGUCGAGACUCGGCAGAUUACCAAGCACGUGG CGCAGAUCUUGGACUCCCGCAUGAACACUAAAUACGAC GAGAACGAUAAGCUCAUCCGGGAAGUGAAGGUGAUUA CCCUGAAAAGCAAACUUGUGUCGGACUUUCGGAAGGA CUUUCAGUUUUACAAAGUGAGAAAUCAACAA cuac AAAUCAGGAAGAGGCCACUGAUCGAAACUAACGGAGA AACGGGCGAAAUCGUGUGGGACAAGGGCAGGGACUUC GCAACUGUUCGCAAAGUGCUCUCUAUGCCGCAAGUCA AUAUUGUGAAGAAAACCGAAGUGCAAACCGGCGGAUU

- 77 047139

UUCAAAGGAAUCGAUCCCCCAAAGAGAAAUAGCGAC AAGCUCAUUGCACGCAAGAAAGACUGGGACCCGAAGA AGUACGGAGGAUUCGAUUCGCCGACUGUCGCAUACUC CGUCCUCGUGGUGGCCAAGGUGGAAGGGAAAGAGC AAAAAGCUCAAAUCCGUCAAAGAGCUGCUGGGGAUUA CCAUCAUGGAACGAUCCUCGUUCGAAGAACCCGAU UGAUUUC CUCGAGGCGAAGGGUUACAAGGAGGUGAAG AAGGAUCUGAUCAUCAAACUCCCAAGUACUCACUGU UCGAACUGGAAAAUGGUCGGAAGCGCAUGCUGGCUUC GGCCGGAGAACUCCAAAAGGAAAUGAGCUGGCCUUG CCUAGCAAGUACGUCAACUUCCUCUAUCUUGCUUCGCA CUACGAAAAACUCAAAGGGUCACCGGAAGAUAACGAA CAGAAGCAGCUUU UCGUGGAGCAGCACAAGCAUUAUC UGGAUGAAAUCAUCGAACAAAUCUCCGAGUUUUCAAA GCGCGUGAUCCUCGCCGACGCCAACCUCGACAAAGUCC UGUCGGCCUACAAUAAGCAUAGAGAUAAGCCGAUCAG AGAACAGGCCGAGAACAUUAUCCACUUGUUCACCCUG ACUAACCUGGGAGCCCAGCCGCCUUCAAGUACUUCGA UACUACUAUCGAUCGCAAAAGAU ACACGUCCACCAAG GAAGUUCUGGACGCGACCCUGAUCCACCAAAGCAUCAC UGGACUCUACGAAACUAGGAUCGAUCUGUCGCAGCUG GGUGGCGAUGGCGGUGGAUCUCCGAAAAAGAAGAGAA AGGUGUAAUGA Amino acid sequence of Cas9 nickase (D10A) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ LFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ EEFYKFIKPILEKMDGTEELLV KLNREDLLRKQRTFDNGSIP HQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER 6

- 78 047139

MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEK LKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGDGGGSPKKKRKV ORF mRNA Cas9 nickases (D10A) AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAA CAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUA CAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAAC ACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAG CACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAAC AAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGA AGAAAGAACAGA AUCUGCUACCUGCAGGAAAUCUUCA GCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAG AAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCG ACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGG CAGACCUGAGACUGAUCUACCUGGCACU GGCACACAUG 7

- 79 047139

AUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACC UGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUC CAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAA ACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUC CUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAA ACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGG ACUGUUCGGAAACCUGA UCGCACUGAGCCUGGGACUG ACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAG ACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGA CGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGU ACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGAC GCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAG AAAUCACAAAGGCACCGCUGAGCGCAAGC AUGAUCAA GAGAUACGACGAACACCACCAGGACCUGACACUGCUGA AGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAA GGAAUCUCUUCGACCAGAGCAAGAACGGAUACGCA GGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCU ACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGG AACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CU GCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCA UCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUC CUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGG ACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAA CAAUCACACCGUGGAA CUUCGAAGAAGUCGUCGACAA GGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCC GAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUAC AACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCA AGACAAACAGAAAG GUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGA AGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGU CGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC

- 80 047139

GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGG ACAACGAAGAAAACGAACAUCCUGGAAGACAUCGU CCUGACACUGACACUGUUCGAAGACAGAGAAUGAUC GAAGAAAGACUGAAGACAUACGCACACCUGUUCGACG ACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACAC AGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGA AUCAGAGACAAGCAGA GCGGAAAGACAAUCCUGGACU UCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAU GCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAA GACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACA GCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCG UCGACGAACUGGUCAAGGUCAUGGGA AGACACAAGCC GGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAG ACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAA UGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGC UGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAA CGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUC GACUGAGCGACUACGACGUCGACCACAUCGUCCC GCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAG GUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCG ACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAA GAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUC ACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGA GAGGAGGACUGAGCGAACU GGACAAGGCAGGAUUCAU CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAG CACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAA AGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAA GGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUC AGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCA ACAACUACCACCACGCACACGACGCAUACCUGAAC GCA GUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGC UGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUA CGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAA

- 81 047139

AUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCA ACAUCAUGAACUUUCAAGACAGAAAUCACACUGGC AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACA AACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAA GAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCC GCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACA GGAGGAUUCAG CAAGGAAAGCAUCCUGCCGAAGAGAA ACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGA CCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGGCAAAGGUCGAAAAGG GAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCU GGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGA UACAAGG AAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUA CAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUG CUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAAC UGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUG GCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAG ACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAA GCACU ACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGG ACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUG UUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAA GUACUUCGACACAACAAUCGACAGAAAGAGAUACACA AGCACAAAGGAAGUCCUG ACGCAACACUGAUCCACCA GAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUG AGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGA AGAAGAGAAAGGUCUAG Amino acid sequence of dCas9 (D10A Н840А) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ LFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG 8

- 82 047139

LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ EEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIP HQIHLGELHAILRRQEDFYPFLK DNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI NNYHHAHDAYLNAVVGTALIKKYPK LESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFKTEITLANGEIRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSFEKNPID FLEAKGYKEV KKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGDGGGSPKKKRK V ORF mRNA dCas9 (D10A H840A) AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAA CAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUA CAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAAC ACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAG CACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAAC AAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGA AGAAAGAACAGA AUCUGCUACCUGCAGGAAAUCUUCA 9

- 83 047139

GCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAG AAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCG ACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGG CAGACCUGAGACUGAUCUACCUGGCACUGGCACACAUG AUCAAGUUCAGAG GACACUUCCUGAUCGAAGGAGACC UGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUC CAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAA ACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUC CUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAA ACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGG ACUGUUCGGAAACCUGAUCGCACUGAGCCUGG GACUG ACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAG ACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGA CGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGU ACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGAC GCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAG AAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAA GACGAACACCACCAGGACCUGACACUGCUGA AGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAA GGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCA GGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCU ACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGG AACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGA GAACAUUCGACAACGGAAGCA UCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUC CUGAGAAGACAGGAACUUCUACCCGUUCCUGAAGG ACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAA CAAUCACACCGUGGAACUUCGAAGAAGUCGUC GACAA GGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCC GAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUAC

- 84 047139

AACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAG GUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGA AGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGU CGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGA AGAUCAUCAAGGACAAGGACUUCCUGG ACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGU CCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUC GAAGAAAGACUGAAGACAUACGCACACCUGUUCGACG ACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACAC AGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGA AUCAGAGACAAGCAGAGCGGAAAGACAAUC G GAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAG ACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAA UGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGC UGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAA CGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUC AACAGACUGAGCGACUAC GACGUCGACGCAAUCGUCCC GCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAG GUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCG ACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAA GAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUC ACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGA GAGGAGGACUGAGCGAACUGGACAAGGCAGGAU UCAU CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAG CACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAA AGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAA

- 85 047139

GGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUC AGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCA ACAACUACCACCACGCACACGACGCAUACCUGAACGCA GUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGC UGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUA CGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAA AUCGGAAAGGCA ACAGCAAAGUACUUCUUCUACAGCA ACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGC AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACA AACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAA GAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCC GCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACA GGAGGAUUCAGCAAGGAAAGCAUC CUGCCGAAGAGAA ACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGA CCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGG GAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCU GGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGG AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUA CAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUG CUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAAC UGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUG GCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAG ACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAA GCACUACCUGGACGAAAUC AUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGG ACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUG UUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAA GUACUUCGACACAACAAUCGACAGAAAGAGAUACACA AGCACAAAGGAAGUCCUGGACGCAACACUGAUC CACCA GAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUG AGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGA AGAAGAGAAAGGUCUAG

- 86 047139

Naked coding follower ity of Cas9 GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAA ACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAA GGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACA GACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAG ACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGA AAGAACAGAAUCUG CUACCUGCAGGAAAUCUUCAGCA ACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAG ACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAG CACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGA AGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACC UGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGA CCUGAGACUGAUCUACCUGGCACUGGCACA CAUGAUCA AGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAA CCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGC UGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUG AGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACC UGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACU GU UCGGAAACCUGAUCGCACUGAGCCUGGGACUGACA CCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGC AAAGCUGCAGCUGAGCAAGGACACAUACGACGACGAC CUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGC AGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCA AUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAA GCUGAGCGCAAGCAUGAUCAAGAG AUACGACGAACACCACCAGGACCUGACACUGCUGAAGG CACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGA AAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGA UACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACA AGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAAC AGAAGAACUGCUGGUCAAGCUGA ACAGAGAAGACCUG CUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCC CGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUG AGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACA 10

- 87 047139

ACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAU CCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCA GAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAU CACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGA GCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACU UCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAA GCACAGCCUGCUGUAC GAAUACUUCACAGUCUACAACG AACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAG AAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCA CAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAU CGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAA GACAGAUUCAACGCAAGCCUGGGAACAU A GACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUG AAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGC UGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAU CCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUG CACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAU CAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGAC GAACUGGUCAAGGUCAU GGGAAGACACAAGCCGGAAA ACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAAC ACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGA UCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAG AACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAA GAGACAUGUACGUCGACCAGGAACUGGACAUCAAC AG ACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGA GCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCU GACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAAC

- 88 047139

GUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACU ACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACA GAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGA GGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGA GACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUAC GACGAAAACGACA AGCUGAUCAGAGAAGUCAAGGUCA UCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAA GGACUUCCAGUUCUACAAGGUCAGAGAAAAUCAACAAC UACCACCACGCACACGACGCAUACCUGAACGCAGUCGU CGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAA AGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACG UCAGAAAGAUGAUCGCAAAGAGCGAACAG GAAAUCGG AAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUC AUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACG GAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGAC UUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGG UCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGG AUUC AGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGC GACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGA AGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUA CAGCGUCCUGGUCGGCAAAGGUCGAAAGGGAAAG AGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAA UCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCC GAUCGACUUCCUGGAAGC AAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCC UGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGC AAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCA CUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAG CCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAAC GAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCA CU ACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAG CAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAG GUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGA

- 89 047139

UCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCAC ACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCA CAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGC AUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCC AGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAA GAGAAAGGUC Naked nickase coding sequence Cas9 GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAA ACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAA GGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACA GACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAG ACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGA AAGAACAGAAUCUG CUACCUGCAGGAAAUCUUCAGCA ACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAG ACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAG CACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGA AGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACC UGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGA CCUGAGACUGAUCUACCUGGCACUGGCACA CAUGAUCA AGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAA CCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGC UGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUG AGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACC UGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACU GU UCGGAAACCUGAUCGCACUGAGCCUGGGACUGACA CCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGC AAAGCUGCAGCUGAGCAAGGACACAUACGACGACGAC CUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGC AGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCA AUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAA GCUGAGCGCAAGCAUGAUCAAGAG AUACGACGAACACCACCAGGACCUGACACUGCUGAAGG CACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGA eleven

- 90 047139

AAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGA UACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACA AGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAAC AGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUG CUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCC CGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUG AGAAG ACAGGAAAGACUUCUACCCGUUCCUGAAGGACA ACAGAGAAAAGAUCGAAAAGAUCCUGAUCAUCAGAAU CCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCA GAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAU CACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGA GCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACU UCGACAAGAACCUGCCGAACGA AAAGGUCCUGCCGAA GCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACG AACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAG AAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCA CAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAU CGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCG AA GACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCU GCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAAC GAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGA CACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGA AAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGA CUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUG AAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGC UGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAU CCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUG CACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAU CAAGAAGGGAAUCCUGCAGACAG UCAAGGUCGUCGAC GAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAA ACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAAC ACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG

- 91 047139

AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGA UCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAG AACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAA GAGACAUGUACGUCGACCAGGAACUGGACAUCAACAG ACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGA GCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCU GCGACAAGAACAGAGGAAAGAGCGACAAC GUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACU ACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACA GAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGA GGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGA GACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAU GAACACAAAGUAC GACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCA UCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAA GGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAAC UACCACCACGCACACGACGCAUACCUGAACGCAGUCGU CGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAA AGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACG UC AGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGG AAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUC AUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACG GAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGAC UUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGG UCAACAUCGUCAAGAAG ACAGAAGUCCAGACAGGAGG AUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGC GACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCGA AGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUA CAGCGUCCUGGUCGGCAAAGGUCGAAAAGGGGAAAG AGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAA UCACAAUCAUGGAAAGAAGCAGCUUCGAAAA GAACCC GAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCC UGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGC

- 92 047139

AAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCA CUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAG CCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAAC GAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACU ACCUGGACCGAAAUCAUCGAACAGAUCAGCGAAUUCAG CAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAG GUCCUGAGCGCAU ACAACAAGCACAGAGACAAGCCGA UCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCAC ACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCA CAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGC AUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCC AGCUGGGAGGAGACGGAGGAGGAAG CCCGAAGAAGAA GAGAAAGGUC Naked coding follower dCas9 GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAA ACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAA GGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACA GACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAG ACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGA AAGAACAGAAUCUG CUACCUGCAGGAAAUCUUCAGCA ACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAG ACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAG CACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGA AGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACC UGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGA CCUGAGACUGAUCUACCUGGCACUGGCACA CAUGAUCA AGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAA CCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGC UGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUG AGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACC UGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACU GU UCGGAAACCUGAUCGCACUGAGCCUGGGACUGACA CCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGC 12

- 93 047139

AAAGCUGCAGCUGAGCAAGGACACAUACGACGACGAC CUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGC AGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCA AUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAA UCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAG AUACGACGAACACCACCAGGACCUGACACUGCUGAAGG CACUG GUCAGACAGCAGCUGCCGGAAAAGUACAAGGA AAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGA UACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACA AGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAAC AGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUG CUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCC CGCACCAGAUCCACCU GGGAGAACUGCACGCAAUCCUG AGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACA ACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAU CCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCA GAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAU CACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGA GCAAGCGCACAGAGCUUCAUCGAAAGAAUGA CAAACU UCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAA GCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACG AACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAG AAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCA CAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAU CGAAU GCUUCGACAGCGUCGAAAUCAGCGGAGUCGAA GACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCU GCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAAC GAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGA CACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGA AAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAG AGAAGAAGUACACAGGAU GGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUG AAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGC

- 94 047139

UGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAU CCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUG CACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAU CAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGAC GAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAA ACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAAC ACAGAAGGGACAGAAGAA CAGCAGAGAAAGAAUGAAG AGAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGA UCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAG AACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAA GAGACAUGUACGUCGACCAGGAACUGGACAUCAACAG ACUGAGCGACUACGACGUCGACGCAAUCGUCCCGCAGA GCUUCCUGAAGGACGACAGCAUCGACAACAA GGUCCU GACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAAC GUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACU ACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACA GAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGA GGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGA GACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU UCCUGGACAGCAGAAUGAACACAAAGUAC GACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCA UCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAA GGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAAC UACCACCACGCACACGACGCAUACCUGAACGCAGUCGU CGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAA AGCGAAUUCGUCUACGGAGACUACA AGGUCUACGACG UCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGG AAAGGCAACAGCAAAGUACUUUCUACAGCAACAUC AUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACG GAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGAC UUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGG U CAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGG AUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGC GACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCGGA

- 95 047139

AGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUA CAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAG AGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAA UCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCC GAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCC UGUUCGAACUGGA AAACGGAAGAAAGAGAAUGCUGGC AAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCA CUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAG CCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAAC GAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACU ACCUGGACCGAAAUCAUCGAACAGAUCAGCGAAUUCAG CAAGAGAGUCAUCCUGGCAGACGCAAACCU GGACAAG GUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGA UCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCAC ACUGAACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCA CAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGC AUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCC GGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAA GAGAAAGGUC Amino acid sequence of Cas9 (without NLS) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ LFEENPINASG VDAKA1LSARLSKSRRLENL1AQLPGEKKNGLFGNL1ALSLG LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ EEFYKFIKPILEKMDGTE ELLVKLNREDLLRKQRTFDNGSIP HQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE 13

- 96 047139

CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQ KGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVI TLKSKLVSDFRKDFQFYKVREI NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFKTEITLANGEIRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR EQAENIIHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGD Cas9 mRNA ORF encoding SEQ ID NO: 13, c usage M codons with minimal uridine content, as indicated in Table 3, with start and stop codons AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAA CAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUA CAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAAC ACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAG CACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAAC AAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGA AGAAAGAACAGA AUCUGCUACCUGCAGGAAAUCUUCA GCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAG AAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCG ACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGG CAGACCUGAGACUGAUCUACCUGGCACU GGCACACAUG AUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACC UGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUC 14

- 97 047139

CAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAA ACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUC CUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAA ACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGG ACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUG ACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAG ACGCAAAGCUGCAGCUGA GCAAGGACACAUACGACGA CGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGU ACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGAC GCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAG AAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAA GAGAUACGACGAACACCACCAGGACCUGACACUGCUGA AGGCACUGGUCAGACAGCAGCUGCCGGA AAAGUACAA GGAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCA GGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUCU ACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGG AACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCA UCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUC CUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGG ACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAA CAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAA GGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAAC CUGCCGAACGAAAAGGUCCUGCC GAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUAC AACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAG GUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGA AGAUCGAAUGCUUCGACAGCGUCG AAAUCAGCGGAGU CGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGG ACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGU

- 98 047139

CCUGACACUGACACUGUUCGAAGACAGAGAAUGAUC GAAGAAAGACUGAAGACAUACGCACACCUGUUCGACG ACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACAC AGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGA AUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACU UCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAU GCAGCUGAUCCACG ACGACAGCCUGACAUUCAAGGAA GACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACA GCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCG UCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCC GGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAG ACAACACAGAGGGACAGAAGAACA GCAGAGAAAGAA UGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGC UGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAA CGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUC AACAGACUGAGCGACUACGACGUCGACCACAUCGUCCC GCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAG G UCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCG ACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAA GAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUC ACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGA GAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAG CACGUCGCACAGAUCCUGG ACAGCAGAAUGAACACAA AGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAA GGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUC AGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCA ACAACUACCACCACGCACACGACGCAUACCUGAACGCA GUCGUCGGAACAGCACUGAUCAAAGUACCCGAAGC UGGAAAGCGAAUUCGUCUACGGAGACUACAA UCUA CGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAA AUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCA ACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGC

- 99 047139

AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACA AACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAA GAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCC GCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACA GGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAA ACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGA CCCGAAGAAGUACGGA GGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGGCAAAGGUCGAAAAGG GAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCU GGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGG AAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUA CAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGA AUG CUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAAC UGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUG GCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAG ACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAA GCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGG GAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUG UUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAA GUACUUCGACACAACAAUCGACAGAAAGAGAUACACA AGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCA GAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUG AGCCAGCUGGGAGGAGACU A.G. Cas9 coding sequence encoding SEQ ID NO: 13, with use of m codons GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAA ACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAA GGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACA GACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAG ACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGA AAGAACAGAAUCUG CUACCUGCAGGAAAUCUUCAGCA ACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAG ACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAG 15

- 100 047139

the minimum uridine content listed in Table 3 (without start or stop codons; suitable for inclusion in the fusion protein coding sequence) CACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGA AGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACC UGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGA CCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCA AGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAA CCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGC UGGUCCAGACA UACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUG AGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACC UGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACU GUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACA CCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGC AAAGCUGCAGCUGAGCAAGGACA CAUACGACGACGAC CUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGC AGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCA AUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAA UCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAG AUACGACGAACACCACCAGGACCUGACACUGCUGAAGG CACUGGUCAGACAGCAGCUGCCGGAAAAGUACA AGA AGACAGGAAGACUUCUACCCGUUCCUGAAGGACA ACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAU CCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCA GAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAU CACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGA GCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACU UCGACAAGAACCUGCCGAAC GAAAAGGUCCUGCCGAA GCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACG AACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAG AAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA

- 101 047139

AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCA CAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAU CGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAA GACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCU GCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAAC GAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGA CACUGACACUGU UCGAAGACAGAGAAAUGAUCGAAGA AAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAU GGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUG AAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGC UGAUCCACGACGACAGCCUGACA UUCAAGGAAGACAU CCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUG CACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAU CAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGAC GAACUGGUCAAGGUCAUGGGAACACAAGCCGGAAA ACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAAC ACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AAGAAGGAAUCAAGGAACUGGGAAGCCAGA UCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAG AACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAA GAGACAUGUACGUCGACCAGGAACUGGACAUCAACAG ACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGA GCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCU GACAAGAAGCGACAAGAA CAGAGGAAAGAGCGACAAC GUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACU ACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACA GAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGA GGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGA GACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGU AC GACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCA UCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAA GGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAAC

- 102 047139

UACCACCACGCACACGACGCAUACCUGAACGCAGUCGU CGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAA AGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACG UCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGG AAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUC AUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACG GAGAAAUC AGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGAC UUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGG UCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGG AUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGC GACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGA AGAAGUACGGAGGAUUCGACAGC CCGACAGUCGCAUA CAGCGUCCUGGUCGGCAAAGGUCGAAAAGGGAAAG AGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAA UCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCC GAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCC UGUUCGAACUGGAAAACGGAAGAAAGAAUGCUGGC A AGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCA CUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAG CCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAAC GACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACU ACCUGGACCGAAAUCAUCGAACAGAUCAGCGAAUUCAG CAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAG GUCCUGAGCGCAUACAA CAAGCACAGAGACAAGCCGA UCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCAC ACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCA CAAAGGAAGUCCUGGACGCAACACUGAUCCACAGAGC AUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCC AGCUGGGAGGAGAC Amino acid successor MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV 16

- 103 047139

nikaseness Cas9 (no NLS) DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI KRYDEHH QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ EEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIP HQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER MTNFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFK EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKL ITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRK RPLIETNGETGEIVWDK GRDFATVRKVLSMPQVNIVKKTE VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGD Cas9 nickase mRNA ORF encoding SEQ ID NO: AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAA CAAACAGCCGUCGGAUGGGCAGUCAUCACAGACGAAUA CAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAAC ACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAG 17

- 104 047139

16, s use of m codons with minimal uridine content as specified in Table 3, with start and stop codons CACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAAC AAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGA AGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUCA GCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAG AAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCG ACGAAGUCGCA UACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGG CAGACCUGAGACUGAUCUACCUGGCACUGGCACACAUG AUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACC UGAACCCGGACAAGCGACGUCGACAAGCUGUUCAUC CAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAA ACCCGAUCAACGCAAGCGGAGUCGAC ACGC AGACCUGUUCCUGGCAGCAAAGAACCUGAGCGAC GCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAG AAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAA GAGAUACGACGAACACCACCAGGACCUGACACUGCUGA AGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAA GGAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCA GGGUACAUCGACGGA AGCAAGCCAGGAAGAAUUCU ACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGG AACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCA UCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUC CUGAGAAGACAGGAACUUCUACCCGUUCCUGAAGG ACAACAGAGAAAAGAUCGAAAAGAUC CUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAA CAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAA

- 105 047139

GGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCC GAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUAC AACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAG GUCAC AGUCAAGCAGCUGAAGGAAGACUACUUCAAGA AGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGU CGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGG ACAACGAAGAAAACGAACAUCCUGGAAGACAUCGU CCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUC GAAGAAAGACUGAAGACAUACGC ACACCUGUUCGACG ACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACAC AGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGA AUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACU UCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAU GCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAA GACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACA GCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCG UCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCC GGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAG ACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAA UGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG GAACACCCGGUCGAAAACACACAGC UGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAA CGGAAGACAUGUACGUCGACCAGGAACUGGACAUC AACAGACUGAGCGACUACGACGUCGACCACAUCGUCCC GCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAG GUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCG ACAACGUCCCGAGCGAAGAAGUCGUCAA GAAGAUGAA GAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUC ACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGA GAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU

- 106 047139

CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAG CACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAA AGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAA GGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUC AGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCA ACAACUACCACCACGCACACGACGCAUACCUGAACGCA GUCGUCGGAAC AGCACUGAUCAAGAAGUACCCGAAGC UGGAAAGCGAAUUCGUCUACGGACUACAAGGUCUA CGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAA AUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCA ACAUCAUGAACUUUCAAGACAGAAAUCACACUGGC AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACA AACGGAGAAACAGGAGAAAUCGUCU GACAAGGGAA GAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCC GCAGGUCAACAUCGUCAAGAACAGAAGUCCAGACA GGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAA ACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGA CCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGUCGUCGCAAAGGUCGAAAAGG GCAAGAAGCUGAAGAGCGUCAAGGAACUGCU GGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGG AAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUA CAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUG CUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAAC UGGCACUGCCGAGCAAGUACG UCAACUUCCUGUACCUG GCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAG ACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAA GCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCCAUCCUGGCAGACGCAAACCUGG ACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUG UUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAA GUACUUCGACACAACAAUCGACAGAAAGAGAUACACA AGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCA

- 107 047139

GAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUG AGCCAGCUGGGAGGAGACUAG Cas9 nickase coding sequence encoding SEQ ID NO: 16, c use of minimum uridine content m codons listed in Table 3 (no start or stop codons; suitable for inclusion in the fusion protein coding sequence) GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAA ACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAA GGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACA GACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAG ACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGA AAGAACAGAAUCUG CUACCUGCAGGAAAUCUUCAGCA ACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAG ACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAG CACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGA AGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACC UGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGA CCUGAGACUGAUCUACCUGGCACUGGCACA CAUGAUCA AGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAA CCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGC UGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUG AGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACC UGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACU GU UCGGAAACCUGAUCGCACUGAGCCUGGGACUGACA CCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGC AAAGCUGCAGCUGAGCAAGGACACAUACGACGACGAC CUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGC AGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCA AUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAA GCUGAGCGCAAGCAUGAUCAAGAG AUACGACGAACACCACCAGGACCUGACACUGCUGAAGG CACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGA AAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGA UACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACA AGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAAC AGAAGAACUGCUGGUCAAGCUGA ACAGAGAAGACCUG CUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCC 18

- 108 047139

CGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUG AGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACA ACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAU CCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCA GAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAU CACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGA GCAAGCGCACA GAGCUUCAUCGAAAGAAUGACAAACU UCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAA GCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACG AACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAG AAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCA CAGUCAAGCAGCUGAAGGAAGAC UACUUCAAGAAGAU CGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAA GACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCU GCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAAC GAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGA CACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGA AAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAU GGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUG AAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGC UGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAU CCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUG CACGAACACAUC GCAAACCUGGCAGGAAGCCCGGCAAU CAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGAC GAACUGGUCAAGGUCAUGGGAAGACAAGCCGGAAA ACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAAC ACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGA UCCUGAAGGAACACCCGGUCGAAAACACACAG CUGCAG AACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAA GAGACAUGUACGUCGACCAGGAACUGGACAUCAACAG ACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGA

- 109 047139

GCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCU GACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAAC GUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACU ACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACA GAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGA GGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGA GACAGCUGGUCG AAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUAC GACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCA UCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAA GGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAAC UACCACCACGCACACGACGCAUACCUGAACGCAGUCGU CGGAACAGCACUGAUCAAGAAGUACCCGA AGCUGGAA AGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACG UCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGG AAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUC AUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACG GAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGAC CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGG UCAACAUCGUCAAAGACAGAAGUCCAGACAGGAGG AUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGC GACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCGA AGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUA CAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAG AGCAAGAAGCUGAAGAGCGUC AAGGAACUGCUGGGAA UCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCC GAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCC UGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGC AAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCA CUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCA AG CCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAAC GAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACU ACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAG

- 110 047139

CAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAG GUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGA UCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCAC ACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCA CAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGC AUCACAGGACU GUACGAAACAAGAAUCGACCUGAGCC AGCUGGGAGGAGAC Amino acid sequence of dCas9 (without NLS) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ LFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ EEFYKFIKPILEKMDGTEELLV KLNREDLLRKQRTFDNGSIP HQIHLGELHAILRRQEDFYPFLKDNREKIEKILFTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQK N SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSK SDFRKDFQFYKVREI NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE 19

- 111 047139

VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKR VILADANLDKVLSAYNKHRDKPIR EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGD dCas9 mRNA ORF encoding SEQ ID NO: 19, c usage M codons with minimal uridine content, as indicated in Table 3, with start and stop codons AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAA CAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUA CAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAAC ACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAG CACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAAC AAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGA AGAAAGAACAGA AUCUGCUACCUGCAGGAAAUCUUCA GCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAG AAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCG ACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGG CAGACCUGAGACUGAUCUACCUGGCACU GGCACACAUG AUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACC UGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUC CAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAA ACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUC CUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAA ACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGG ACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUG ACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAG ACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGA CGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGU ACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGAC GCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAG AAAUCACAAAGGCACCGCU GAGCGCAAGCAUGAUCAA GAGAUACGACGAACACCACCAGGACCUGACACUGCUGA AGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAA 20

- 112 047139

GGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCA GGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCU ACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGG AACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCA UCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUC CUGA GAAGACAGGAAGACUUCUACCCGUUCCUGAAGG ACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAA CAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAA GGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCC GAACGAAAAGGUCCUGCC GAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUAC AACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAG GUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGA AGAUCGAAUGCUUCGACAGCGUCGAAAUCAG CGGAGU CGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGGAAGAUCAUCAAGGACAAGGACUUCCUGG ACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGU CCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUC GAAGAAAGACUGAAGACAUACGCACACCUGUUCGACG ACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACGGAA GACUGAGCAGAAAGCUGAUCAACGGA AUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACU UCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAU GCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAA GACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACA GCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUG CAGACAGUCAAGGUCG UCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCC GGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAG ACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAA

- 113 047139

UGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGC UGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAA CGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUC AACAGACUGAGCGACUACGACGUCGACGCAAUCGUCCC GCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAG GUCCUGACAAG AAGCGACAAGAACAGAGGAAAGAGCG ACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAA GAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUC ACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGA GAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAG CACGUCGCACAGAUCCUGGACAGCAGAAU GAACACAA AGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAA GGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUC AGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCA ACAACUACCACCACGCACACGACGCAUACCUGAACGCA GUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGC UGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUA UCAGAAAGAUGAUCGCAAAGAGCGAACAGGAA AUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCA ACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGC AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACA AACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAA GAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCC GCAGGUCAACAUCGUCAAG AAGACAGAAGUCCAGACA GGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAA ACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGA CCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGGCAAAGGUCGAAAAGG GAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCU GGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGG AAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUA CAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUG

- 114 047139

CUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAAC UGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUG GCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAG ACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAA GCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGG ACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUG UUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAA GUACUUCGACACAACAAUCGACAGAAAGAGAUACACA AGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCA GAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUG AGCCAGCUGGGAGGAGACUAG dCas9 coding sequence encoding SEQ ID NO: 19, with use of minimum uridine content m codons listed in Table 3 (no start or stop codons; suitable for inclusion in the fusion protein coding sequence) GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAA ACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAA GGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACA GACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAG ACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGA AAGAACAGAAUCUG CUACCUGCAGGAAAUCUUCAGCA ACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAG ACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAG CACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGA AGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACC UGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGA CCUGAGACUGAUCUACCUGGCACUGGCACA CAUGAUCA AGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAA CCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGC UGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUG AGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACC UGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACU GU UCGGAAACCUGAUCGCACUGAGCCUGGGACUGACA CCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGC AAAGCUGCAGCUGAGCAAGGACACAUACGACGACGAC 21

- 115 047139

CUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGC AGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCA AUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAA UCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAG AUACGACGAACACCACCAGGACCUGACACUGCUGAAGG CACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGA AAUCUU CUUCGACCAGAGCAAGAACGGAUACGCAGGA UACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACA AGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAAC AGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUG CUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCC CGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUG AGAAGACAGGAAGA CUUCUACCCGUUCCUGAAGGACA ACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAU CCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCA GAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAU CACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGA GCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACU UCGACAAGAACCUGCCGAACGAAAAGGUCCUGC CGAA GCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACG AACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAG AAAGCCGGCAUUCCUGAGCGGAACAGAAGAAGGCA AUCGUACCUGCUGUUCAAGACAAACAGAAAGGUCA CAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAU CGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAA UCAACGCAAGCCUGGGAACAUACCACGACCU GCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAAC GAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGA CACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGA AAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAU GGGGAAGACUGAGCAGAAA GCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUG AAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGC UGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAU

- 116 047139

CCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUG CACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAU CAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGAC GAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAA ACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAAC ACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAG GAACUGGGAAGCCAGA UCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAG AACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAA GAGACAUGUACGUCGACCAGGAACUGGACAUCAACAG ACUGAGCGACUACGACGUCGACGCAAUCGUCCCGCAGA GCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCU GACAAGAAGCGACAAGAACAGAGGAAAGAGCGA CAAC GUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACU ACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACA GAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGA GGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGA GACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUAC GACGAAAACGA CAAGCUGAUCAGAGAAGUCAAGGUCA UCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAA GGACUUCCAGUUCUACAAGGUCAGAGAAAAUCAACAAC UACCACCACGCACACGACGCAUACCUGAACGCAGUCGU CGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAA AGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACG UCAGAAAGAUGAUCGCAAAGAGCGAAC AGGAAAUCGG AAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUC AUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACG GAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGAC UUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGG UCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGG AU UCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGC GACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGA AGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUA

- 117 047139

CAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAG AGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAA UCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCC GAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCC UGUUCGAACUGGAAAACGGAAGAAAGAAUGCUGGC AAGCGCAGGAGAA CUGCAGAAGGGAAACGAACUGGCA CUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAG CCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAAC GAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACU ACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAG CAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAG GUCCUGAGCGCAUACAACAAGCACAGAGA CAAGCCGA UCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCAC ACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCA CAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGC AUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCC AGCUGGGAGGAGACGGAGGAGGAAGC Amino acid sequence of Cas9 with two nuclear localization signals as terminal amino acids MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ LFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ EEFYKFIKPILEKMDGTEELLV KLNREDLLRKQRTFDNGSIP HQIHLGELHAILRRQEDFYPFLKDNREKIEKILFTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR 22

- 118 047139

YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVP QSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLA SAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGD GSGSPKKKRKVDGSPKKKRKVDSG Cas9 mRNA ORF encoding SEQ ID NO: 22, c use of m codons with minimal uridine content as specified in Table 3, with start and stop codons AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAA CAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUA CAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAAC ACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAG CACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAAC AAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGA AGAAAGAACAGA AUCUGCUACCUGCAGGAAAUCUUCA GCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAG AAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCG ACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGG CAGACCUGAGACUGAUCUACCUGGCACU GGCACACAUG AUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACC UGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUC CAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAA 23

- 119 047139

ACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUC CUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAA ACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGG ACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUG ACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAG ACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGA CGACCUGGACAACCU GCUGGCACAGAUCGGAGACCAGU ACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGAC GCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAG AAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAA GAGAUACGACGAACACCACCAGGACCUGACACUGCUGA AGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAA GGGAAUCUUCUUCGACCAGAGCAAGAA CGGAUACGCA GGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCU ACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGG AACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCA UCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUC CUGAGAAGACAGGAAGACUUCUACCCGUUCCUGA AGG ACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAA CAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAA GGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCC GAAGCACAGCCU GCUGUACGAAUACUUCACAGUCUAC AACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGGUUCAAGACAAACAGAAAG GUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGA AGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGU CGAAGACAGAUUCAACGCAAGCCU GGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGG ACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGU CCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUC

- 120 047139

GAAGAAAGACUGAAGACAUACGCACACCUGUUCGACG ACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACAC AGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGA AUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACU UCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAU GCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAA GACAUCCAGAAGG CACAGGUCAGCGGACAGGGAGACA GCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCG UCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCC GGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAG ACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAA UGAAGAGAAUCGAAGAAGGAAUCAA GGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGC UGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAA CGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUC AACAGACUGAGCGACUACGACGUCGACCACAUCGUCCC GCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAG GUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCG AC AACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAA GAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUC ACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGA GAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAG CACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAA AGUACGACGAAAACGACAAG CUGAUCAGAGAAGUCAA GGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUC AGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCA ACAACUACCACCACGCACACGACGCAUACCUGAACGCA GUCGUCGGAACAGCACUGAUCAAAGUACCCGAAGC UGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUA CGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAG GAA AUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCA ACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGC AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACA

- 121 047139

AACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAA GAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCC GCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACA GGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAA ACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGA CCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCU GGUCGUCGCAAAGGUCGAAAAGG GAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCU GGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGG AAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUA CAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUG CUGGCAAGCGCAGGAGAACUGCAGAAGGGAAA CGAAC UGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUG GCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAG ACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAA GCACUACCUGGACGAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCCAUCCUGGCAGACGCAAACCUGG ACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAG AGAACAGGCAGAAAACAUCAUCCACCUG UUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAA GUACUUCGACACAACAAUCGACAGAAAGAGAUACACA AGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCA GAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUG AGCCAGCUGGGAGGAGACGGAAGCGGAAGCCCGAAGA AGAAGAGAAAGGUCGACGGA AGCCCGAAGAAGAAGAG AAAGGUCGACAGCGGAUAG Cas9- coding follower ness, coding SEQ ID NO: 23, s usage GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAA ACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAA GGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACA GACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAG ACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGA AAGAACAGAAUCUG CUACCUGCAGGAAAUCUUCAGCA ACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAG 24

- 122 047139

m codons with minimal uridine content listed in Table 3 (no start or stop codons; suitable for inclusion in the fusion protein coding sequence) ACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAG CACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGA AGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACC UGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGA CCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCA AGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAA CCCGGACAACA GCGACGUCGACAAGCUGUUCAUCCAGC UGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUG AGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACC UGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACU GUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACA CCGAACUUCAAGAGCAACUUCGAC CUGGCAGAAGACGC AAAGCUGCAGCUGAGCAAGGACACAUACGACGACGAC CUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGC AGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCA AUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAA UCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAG AUACGACGAACACCACGGACCUGACACUGCU GAAGG CACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGA AAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGA UACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACA AGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAAC AGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUG CUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCC CGCA CCAGAUCCACCUGGGAGAACUGCACGCAAUCCUG AGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACA ACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAU CCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCA GAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAU CACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGA GCAAGCGCACAGAGCUUC AUCGAAAGAAUGACAAACU UCGACAAGACCUGCCGAACGAAAAGGUCCUGCCGAA GCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACG AACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAG

- 123 047139

AAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCA CAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAU CGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAA GACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCU GCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAAC GAAGAAAACGAA GACAUCCUGGAAGACAUCGUCCUGA CACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGA AAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAU GGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUG AAGAGCGACGGAUUCGCAAACAG AAACUUCAUGCAGC UGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAU CCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUG CACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAU CAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGAC GAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAA ACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAAC GGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGA UCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAG AACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAA GAGACAUGUACGUCGACCAGGAACUGGACAUCAACAG ACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGA GCUUCCUGAAGGACGACAG CAUCGACAACAAGGUCCU GACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAAC GUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACU ACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACA GAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGA GGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGA GACAGCUGGUCGAAACAAGACAGAUCACAAAGCAC GU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUAC GACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCA UCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAA

- 124 047139

GGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAAC UACCACCACGCACACGACGCAUACCUGAACGCAGUCGU CGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAA AGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACG UCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGG AAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUC AUGAACUU CUUCAAGACAGAAAUCACACUGGCAAACG GAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGAC UUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGG UCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGG AUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGC GACAAGCUGAUCGCAAGAAAGAAG GACUGGGACCCGA AGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUA CAGCGUCCUGGUCGGCAAAGGUCGAAAAGGGAAAG AGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAA UCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCC GAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCC UGU UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGC AAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCA CUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAG CCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAAC GAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACU ACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAG CAAGAGUCAUCCUGGCA GACGCAAACCUGGACAAG GUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGA UCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCAC ACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCA CAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGC AUCACAGGACUGUACGAAACAAGAAUCGACCU GAGCC AGCUGGGAGGAGACGGAAGCGGAAGCCCGAAGAAGAA GAGAAAGGUCGACGGAAGCCCCGAAGAAGAAGAGAAAG GUCGACAGCGGA

- 125 047139

Amino acid sequence of Cas9 nickase with two nuclear localization signals as terminal amino acids MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ LFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ EEFYKFIKPILEKMDGTEELLV KLNREDLLRKQRTFDNGSIP HQIHLGELHAILRRQEDFYPFLKDNREKIEKILFTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQK N SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSK SDFRKDFQFYKVREI NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFKTEITLANGEIRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV AYSVLV VAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR EQAENIIHLFTLTNLGAPAAFKYFDTTI DRKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGDGSGSPKKKRKVDGSPKKK RKVDSG 25

- 126 047139

Cas9 nickase mRNA ORF encoding SEQ ID NO: 25, c usage M codons with minimal uridine content, as indicated in Table 3, with start and stop codons AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAA CAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUA CAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAAC ACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAG CACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAAC AAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGA AGAAAGAACAGA AUCUGCUACCUGCAGGAAAUCUUCA GCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAG AAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCG ACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGG CAGACCUGAGACUGAUCUACCUGGCACU GGCACACAUG AUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACC UGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUC CAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAA ACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUC CUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAA ACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGG ACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUG ACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAG ACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGA CGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGU ACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGAC GCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAG AAAUCACAAAGGCACCGCU GAGCGCAAGCAUGAUCAA GAGAUACGACGAACACCACCAGGACCUGACACUGCUGA AGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAA GGAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCA GGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCU ACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGG AACAGAAGAACUGCUGGUCAAGCUGAACAGA GAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCA UCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUC CUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGG 26

- 127 047139

ACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAA CAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAA GGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCC GAAGCACAGCCUG CUGUACGAAUACUUCACAGUCUAC AACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGGUUCAAGACAAACAGAAAG GUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGA AGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGU CGAAGACAGAUUCAACGCAAGCCU GAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGG ACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGU CCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUC GAAGAAAGACUGAAGACAUACGCACACCUGUUCGACG ACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACAC AGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGA AUCAGA A GGUCAUGGGAAGACACAAGCC GGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAG ACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAA UGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGC UGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAA CGGAAGACAUGUACGUCGACCAGGAACUGGA CAUC AACAGACUGAGCGACUACGACGUCGACCACAUCGUCCC GCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAG GUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCG

- 128 047139

ACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAA GAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUC ACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGA GAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAG CACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAA AGUACGACGAAAACGA CAAGCUGAUCAGAGAAGUCAA GGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUC AGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCA ACAACUACCACCACGCACACGACGCAUACCUGAACGCA GUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGC UGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUA CGACGUCAGAAAGAUGAUCGCAAAGAGCGA ACAGGAA AUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCA ACAUCAUGAACUUUCAAGACAGAAAUCACACUGGC AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACA AACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAA GAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCC GCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACA AUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAA ACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGA CCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGG GAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCU GGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAG AACCCGAUCGACUUCCUGGAAGC AAAGGGAUACAAGG AAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUA CAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUG CUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAAC UGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUG GCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAG ACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAA GCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGG ACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA

- 129 047139

GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUG UUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAA GUACUUCGACACAACAAUCGACAGAAAGAGAUACACA AGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCA GAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUG AGCCAGCUGGGAGGAGACGGAAGCGGAAGCCCGAAGA AAAGGUCGACGGAAGCCCCGAAGAAGAAGAG AAAGGUCGACAGCGGAUAG Cas9 nickase coding sequence encoding SEQ ID NO: 25, s use of minimum uridine content m codons listed in Table 3 (no start or stop codons; suitable for inclusion in the fusion protein coding sequence) GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAA ACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAA GGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACA GACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAG ACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGA AAGAACAGAAUCUG CUACCUGCAGGAAAUCUUCAGCA ACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAG ACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAG CACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGA AGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACC UGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGA CCUGAGACUGAUCUACCUGGCACUGGCACA CAUGAUCA AGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAA CCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGC UGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUG AGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACC UGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACU GU UCGGAAACCUGAUCGCACUGAGCCUGGGACUGACA CCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGC AAAGCUGCAGCUGAGCAAGGACACAUACGACGACGAC CUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGC AGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCA AUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAA GCUGAGCGCAAGCAUGAUCAAGAG AUACGACGAACACCACCAGGACCUGACACUGCUGAAGG 27

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CACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGA AAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGA UACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACA AGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAAC AGAAGAACUGCUGGUCAAGCUGAACAGAAGACCUG CUGAGAAAGCAGAGAACAUUCCAACGGAAGCAUCC CGCACCAG AUCCACCUGGGAGAACUGCACGCAAUCCUG AGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACA ACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAU CCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCA GAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAU CACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGA GCAAGCGCACAGAGCUUCAUCG AAAGAAUGACAAACU UCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAA GCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACG AACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAG AAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCA CAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAG au GCUGAAGAGAAGAAGAUACACAGGAU GGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUG AAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGC UGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAU CCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUG CACGAACACAUCGCAAACCUGGCA GGAAGCCCGGCAAU CAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGAC GAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAA ACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAAC

- 131 047139

ACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGA UCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAG AACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAA GAGACAUGUACGUCGACCAGGAACUGGACAUCAACAG ACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGA GCUUCCUGAAG GACGACAGCAUCGACAACAAGGUCCU GACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAAC GUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACU ACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACA GAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGA GGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGA GACAGCUGGUCGAAACAAGACAGAUC ACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUAC GACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCA UCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAA GGACUUCCAGUUCUACAAGGUCAGAGAAAAUCAACAAC UACCACCACGCACACGACGCAUACCUGAACGCAGUCGU CGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAA AGCGA AUUCGUCUACGGAGACUACAAGGUCUACGACG UCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGG AAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUC AUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACG GAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGAC UUCGCAACAGUCAGAAA UCCUGAGCAUGCCGCAGG UCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGG AUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGC GACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCGGA AGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUA CAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAG AGCAAGAAGCUGAAGAGCGGGAAGGAACUGCU GAA UCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCC GAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCC

- 132 047139

UGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGC AAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCA CUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAG CCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAAC GAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACU ACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAG CAAGAGAGUCAUC CUGGCAGACGCAAACCUGGACAAG GUCCUGAGCGCAUACAACAAGCACAGACAAGCCGA UCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCAC ACUGAACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCA CAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGC AUCACAGGACUGUACGAAACAAGAAUC GACCUGAGCC AGCUGGGAGGAGAC GGAAGCGGAAGCCCCGAAGAAGAAGAGAAAGGUCGACG GAAGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGA Amino acid sequence of dCas9 with two nuclear localization signals as terminal amino acids MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ LFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ EEFYKFIKPILEKMDGTEELLV KLNREDLLRKQRTFDNGSIP HQ1HLGELHA1LRRQEDFYPFLKDNREK1EK1LTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG QKN 28

- 133 047139

SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSKLV SDFRKDFQFYKVREI NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFKTEITLANGEIRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV AYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR EQAENIIHLFTLTNLGAPAAFKYFDTTIDR KRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGDGSGSPKKKRKVDGSPKKK RKVDSG dCas9 mRNA ORF encoding SEQ ID NO: 28, c usage M codons with minimal uridine content, as indicated in Table 3, with start and stop codons AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAA CAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUA CAAGGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAAC ACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAG CACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAAC AAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGA AGAAAGAACAGA AUCUGCUACCUGCAGGAAAUCUUCA GCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAG AAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCG ACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGG CAGACCUGAGACUGAUCUACCUGGCACU GGCACACAUG AUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACC UGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUC CAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAA ACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUC CUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAA ACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGG 29

- 134 047139

ACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUG ACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAG ACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGA CGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGU ACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGAC GCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAG AAAUCACAAA GGCACCGCUGAGCGCAAGCAUGAUCAA GAGAUACGACGAACACCACCAGGACCUGACACUGCUGA AGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAA GGAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCA GGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCU ACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGG AACAGAAGAACUGCUGGUCAAG CUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCA UCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUC CUGAGAAGACAGGAACUUCUACCCGUUCCUGAAGG ACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGA AGAAA CAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAA GGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCC GAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUAC AACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA UCGUCGACCUGCUGUUCAAGACAAACAGAAAG GUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGA AGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGU CGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGG ACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGU CCUGACACUGACACUGUUCGA AGACAGAGAAAUGAUC GAAGAAAGACUGAAGACAUACGCACACCUGUUCGACG ACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACAC AGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGA

- 135 047139

AUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACU UCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAU GCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAA GACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACA GCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCG UCGACG AACUGGUCAAGGUCAUGGGAAGACACAAGCC GGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAG ACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAA UGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGC UGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAA CGGAAGACAUGUACGUCGACCA GGAACUGGACAUC AACAGACUGAGCGACUACGACGUCGACGCAAUCGUCCC GCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAG GUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCG ACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAA GAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUC ACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGA GAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAG CACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAA AGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAA GGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUC AGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCA ACAACUACCACCACG CACACGACGCAUACCUGAACGCA GUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGC UGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUA CGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAA AUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCA ACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGC AAACGGAGAAAUCAGAAAGAGACCGCU GAUCGAAACA AACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAA GAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCC GCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACA

- 136 047139

GGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAA ACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGA CCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGG GAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCU GGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAG AACCCGAUCGACUUC CUGGAAGCAAAGGGAUACAAGG AAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUA CAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUG CUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAAC UGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUG GCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAG ACAACGAACAGAAGCAGCUGUUCGUCGAA CAGCACAA GCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGG ACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUG UUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAA GUACUUCGACACAACAAUCGACAGAAAGAGAUACACA AGCAC AAAGGAAGUCCUGGACGCAACACUGAUCCACCA GAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUG AGCCAGCUGGGAGGAGAC GGAAGCGGAAGCCCCGAAGAAGAAGAGAAAGGUCGACG GAAGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGAUA G dCas9 coding sequence encoding SEQ ID NO: 28, with use of m codons with minimal GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAA ACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAA GGUCCCGAGCAAGAAGUUCAAGGUCCUGGGAAACACA GACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAG ACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGA AAGAACAGAAUCUG CUACCUGCAGGAAAUCUUCAGCA ACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAG ACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAG CACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGA thirty

- 137 047139

uridine content listed in Table 3 (without start or stop codons; suitable for inclusion in the fusion protein coding sequence) AGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACC UGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGA CCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCA AGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAA CCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGC UGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC CAAGCGGAGUCGACGCAAAGGCAAUCCUG AGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACC UGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACU GUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACA CCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGC AAAGCUGCAGCUGAGCAAGGACACAUACGACGACGAC CUGGACAACCUGCUGGCA CAGAUCGGAGACCAGUACGC AGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCA AUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAA UCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAG AUACGACGAACACCACCAGGACCUGACACUGCUGAAGG CACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGA AAUCUUCUUCGACCAGCAAGAACGGAU ACGCAGGA UACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACA AGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAAC AGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUG CUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCC CGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUG AGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACA ACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAU CCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCA GAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAU CACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGA GCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACU UCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAA GCACAGCCUGCUGUACGA AUACUUCACAGUCUACAACG AACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAG AAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCA

- 138 047139

CAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAU CGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAA GACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCU GCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAAC GAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGA CACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGA AAGACUGAAGAC AUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAU GGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUG AAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGC UGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAU CCAGAAGGCACAGGUCAGCGGACA GGGAGACAGCCUG CACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAU CAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGAC GAACUGGUCAAGGUCAUGGAAGACACAAGCCGGAAA ACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAAC ACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGA AGGAACACCCGGUCGAAAACACACAGCUGCAG AACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAA GAGACAUGUACGUCGACCAGGAACUGGACAUCAACAG ACUGAGCGACUACGACGUCGACGCAAUCGUCCCGCAGA GCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCU GACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAAC GUCCCGAGCGAAGAAGUCGUC AAGAAGAUGAAGAACU ACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACA GAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGA GGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGA GACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUAC GACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCA UCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAA GGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAAC UACCACCACGCACACGACGCAUACCUGAACGCAGUCGU

- 139 047139

CGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAA AGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACG UCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGG AAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUC AUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACG GAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGG AGAAAUCGUCUGGGACAAGGGAAGAGAC UUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGG UCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGG AUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGC GACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCG AGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUA CAGCGUCCUGGUCGUCGCAAAUC GAAAAGGGAAAG AGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAA UCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCC GAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCC UGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGC AAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCA CU GCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAG CCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAAC GAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACU ACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAG CAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAG GUCCUGAGCGCAUACAACAAGCACAGACAAGCCGA UCAGAGAACAGGCAGAAAA CAUCAUCCACCUGUUCAC ACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCA CAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGC AUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCC AGCUGGGAGGAGAC GGAAGCGGAAGCCCCGAAGAAGAAGAGAAAGGUCGACG GAAGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGA T7 promoter TAATACGACTCACTATA 31 5'-UTR ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTC 32

- 140 047139

human beta globin AAACAGACACC Z'-UTR of human beta-globin GCTCGCTTTCTTGCTGTCCAATTTTCTATTAAAGGTTCCTT TGTTCCCTAAGTCCAACTACTACTAAACTGGGGGATATTATG AAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACA TTTATTTTCATTGC 33 5'-UTR of human alphaglobin CATAAACCCTGGCGCGCTCGCGGCCCGGCACTCTTCTGG TCCCCACAGACTCAGAGAGAACCCACC 34 Z'-UTR of human alphaglobin GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCC TCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC GTGGTCTTTGAATAAAGTCTGAGTGGGCGGC 35 5'-UTR of Xenopus laevis betaglobin AAGCTCAGAATAAAACGCTCAACTTTGGCC 36 Z'-UTR of betaglobin Xenopus laevis ACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTAC ATAATACCAACTTACACTTTACAAAATGTTGTCCCCCAA AATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGT TTCTTCACATTCT 37 5'-UTR growth hormone large cattle CAGGGTCCTGTGGACAGCTCACCAGCT 38 Z'-NTO of bovine growth hormone TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCC TTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAAT AAAATGAGGAAATTGCATCGCA 39 Z'-UTR alpha, mature chain 1 of hemoglobin Mus musculus (Hba-al) GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCT TCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAG CCTGAGTAGGAAG 40

- 141 047139

5'-HTO HSD17B4 TCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTG TGTGTCGTTGCAGCCTTATTC 41 Guide RNA G282 targeting TTR mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmC mUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCU AGU SS GUU AU C AmAmCmU mU mGmAmAmAmAmAmGmU mGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU *mU*mU*mU 42 Cas9 transcript with 5'-UTR HSD, ORF corresponding to SEQ ID NO: 4, Kozak sequence and Z'-NTO ALB GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGT GTGTGTGTCGTTGCAGGCCTTATTCGGATCCGCCACCAT GGACAAGAAGTACAGCATCGGACTGGACATCGGAACAA ACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAG GTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGA CAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGC GACAGCGGAGAAACAGCAGAAGCAACAAGACTG AAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGA ACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAA ATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGA AGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAA GACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCAT ACCACGAAAAGTACCCCGACAAT CTACCACCTGAGAAAG AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACT GATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGG ACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACA GCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACAT ACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGC GGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGA G CAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGC CGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATC GCACTGAGCCTGGACTGACACCGAACTTCAAGAGCAA CTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCA AGGACACATACGACGACGACCTGGACAACCTGCTGGCA CAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGC AAAGAACCTGAGCGA CGCAATCCTGCTGAGCGACATCC TGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGC GCAAGCATGATCAAGAGATACGACGAACACCACCAGGA 43

- 142 047139

CCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCC GGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGA ACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAG GAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACA GAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAAC GGAAGCATCCCGCACC AGATCCACCTGGGAGAACTGCA CGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCT GAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACAT TCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGA AACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGA AACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACA AGGGAGCAAGCGCACAGAGCTTCATCGAAAGA AATGCTT CGACAGCGTCGAAATCAGCGGAGTCGAAGAC AGATTCAACGCAAGCCTGGGAACATACCACGACCTGCT GAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAG AAAACGAAGACATCCTGGAAGACATCGTCCTGACACTG ACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACT GAAGACATACGCACACCTGTTCGACGACAAGGTCATGA AGCAGCTGAAGAGAAGAAGATA CACAGGATGGGGAAG ACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGC AGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGAC GGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGAC GACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACA GGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCG CAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATC CTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGT CATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAA TGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAA

- 143 047139

GAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGA ATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCC GGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACC TGTACTACCTGCAGAACGGAAGAGACATGTACGTCGAC CAGGAACTGGACATCAACAGACTGAGCGACTACGACGT CGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACA GCATCGACAAC AAGGTCCTGACAAGAAGCGACAAGAAC AGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGT CAAGAAGATGAAACTACTGGAGACAGCTGCTGAACG CAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACA AAGGCAGAGAGGAGGACTGAGCGAACTGGACAAGG CAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAG ATCACAAAGCACGTCGCACAGATCCT GGACAGCAGAAT GAACACAAAGTACGACGAAAACGACAAGCTGATCAGAG AAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGC GACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGA AATCAACAACTACCACCACGCACACGACGCATACCTGA ACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCG AAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGT CTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGG AAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGC AACATCATGAACTTCTTCAAGACAGAAATCACACTGGCA AACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAA CGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGA GACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCA GGTCAACATCGTCAAGAAGA CAGAAGTCCAGACAGGAG GATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGC GACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACA GCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGC AAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCAC AATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCG ACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAG GACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAA CTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG

- 144 047139

AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCA AGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAA AGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCA GCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAAT CATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCC TGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATAC ACAGAGACAAGCCGATCAGAGAACAGGCAG AAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCA ACACTGATCCACCAGAGCATCACAGGACTGTACGAAAC AAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAG GAAGCCCGAAGAAGAAGAGAAA TCTAGCTAGCCATC ACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAG AAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTTC TTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAAAC ATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTC AATTAATAAAAAAATGGAAAGAACCTCGAG Cas9 transcript with 5'-UTR HSD, ORF corresponding to SEQ ID NO: 4, and 3'UTR ALB GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGT GTGTGTGTCGTTGCAGGCCTTATTCGGATCCATGGACAA GAAGTACAGCATCGGACTGGACATCGGAACAAACAGCG TCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCG AGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACA CAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCG ACA GCGGAGAAACAGCAGAAGCAACAAGACTGAAGAG AACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGA ATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCA AAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAG CTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACC CGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACG AAAAGTACCCGACAATCT ACCACCTGAGAAAGAAGCTG GTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTA CCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTT CCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACG TCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACC 44

- 145 047139

AGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTC GACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAG CAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAG AAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGA CCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACA CATACGACGACGACCTGG ACAACCTGCTGGCACAGATC GGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAA CCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGT CAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCA TGATCAAGAGATACGACGAACACCACCAGGACCTGACA CTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAA GTACAAGGAAATCTTCTTCGACCAGAGCAAGAAC GGAT ACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAA TTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGAC GGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGA CCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCA TCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCC TGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC GATCGAAAAGATCCTGACATTCAGAAT CCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCA GATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATC ACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGCTTCATCGAAAGAATGACAAACTTCG ACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCAC AGCCTGCTGTACGAATACTTCACAGTC TACAACGAACTG ACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCG ACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAG CAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTT CGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCA ACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATC AT CAAGGACAAGGACTTCCTGGACAACGAAGAAAACGA AGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACAT

- 146 047139

ACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTG AAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCA GAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGA AAGACAATCTGGACTTCCTGAAGAGCGACGGATTCGC AAACAGAAACTTCATGCAGCTGATCCACGACGACAGCC TGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGC GGACAGGGAGACA GCCTGCACGAACACATCGCAAACCT GGCAGGAAGCCCGCAATCAAGAAGGGAATCCTGCAGA CAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGA AGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAG AGAAAACCAGACAACACAGAAGGGACAGAAGAACAGC AGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGG AACTGGGAAGCCAGATCTGAAGGAAC ACCCGGTCGAA AACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTA CCTGCAGAACGGAAGACATGTACGTCGACCAGGAAC TGGACATCAACAGACTGAGCGACTACGACGTCGACCAC ATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGAC AACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAA AGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAG A TGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCT GATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAG AGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTC ATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAA GCACGTCGCACAGATCCTGGACAGCAGAATGAACACAA AGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAG GTCATCACACTGAAG CAAGCTGGTCAGCGACTTCAG AAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACA ACTACCACCACGCACACGACGCATACCTGAACGCAGTC GTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGA AAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGT CAGAAAGATGATCGCAAAGAGCGAACAGAAATCGGAA AGGCAACAGCAAAGTACTTCTTCTACAGCAA CATCATGA ACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAA ATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAAC AGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAA

- 147 047139

CAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATC GTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAA GGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGA TCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGA GGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTC GTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGA AGACGTCAAGGAACT GCTGGGAATCACAATCATGGAA AGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGA AGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCA TCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAAC GGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCA GAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCA ACTTCCTGTACCTGGCAAGCCACTACGAAAAGC TGAAGG GAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTC GAACAGCACAAGCACTACCTGGACGAAATCATCGAACA GATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACG CAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCAC AGAGACAAGCCGATCAGAACAGGCAGAAAACATCAT CCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGC ATTCAAGT ACTTCGACACAACAATCGACAGAAAGAGAT ACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATC CACCAGAGCATCACAGGACTGTACGAAACAAGAATCGA CCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGA AGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAA AGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAAT GAAGATCAATAGCTTATTCATCTCTTTTTCTTT TTCGTTG GTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCT TTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAA AAAATGGAAAGAACCTCGAG Alternative Cas9 ORF with 19.36% U content ATGGATAAGAAGTACTCGATCGGGCTGGATATCGGAAC TAATTCCGTGGGTTGGGCAGTGATCACGGATGAATACAA AGTGCCGTCCAAGAAGTTCAAGGTCCTGGGGAACACCG ATAGACACAGCATCAAGAAGAATCTCATCGGAGCCCTG CTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTC AAACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAA 45

- 148 047139

TCGCATCTGCTATCTGCAAGAAATCTTTTCGAACGAAAT GGCAAAGGTGGACGACAGCTTCTTCCACCGCCTGGAAG AATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGG CATCCTATCTTTGGAAACATCGTGGACGAAGTGGCGTAC CACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAA GTTGGTTGACTCAACTGACAAGGCCGACCTCAGATTGAT CTACTTGGCCCTC GCCCATATGATCAAATTCCGCGGACA CTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGA CGTGGATAAGCTGTTCATTCAACTGGTGCAGACCTACAA CCAACTGTTCGAAGAAAACCCAATCAATGCCAGCGGCG TCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGT CGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGA GAGAAGAAGAACGGACTTTTCGGCA ACTTGATCGCTCTC TCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGAC CTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACAC CTACGACGACGACTTGGACAATTTGCTGGCACAAATTGG CGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCT TTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAA CACCGAAATAACCAAAGCGCCGCTTAGCGCTCGATG AT TAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCT CAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACA AGGAGATTTTCTTCGACCAGTCCAAGAATGGGTACGCAG GGTACATCGATGGAGGCGCCAGCCAGGAAGAGTTCTAT AAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAAC CGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGC CAGAGAACCTTTGACAACGGAAGCATTCCA CACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGG CGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGG GAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTAT TACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCG TGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTG GAATTTCGAGGAAGTT GTGGATAAGGGAGCTTCGGCAC AATCCTTCATCGAACGAATGACCAACTTCGACAAGAATC TCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTT ACGAATACTTCACTGTCTACAACGAACTGACTAAAGTGA

- 149 047139

AATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGA GCGGAGAACAGAAGAAAGCGATTGTCGATCTGCTGTTC AAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGA GGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGA AATCAGCGGAGTGGAGGACAGATTCAACGCTTCGCTGG GAACCTATCATGATCTCCTGAAGATCATCAAGGACAAGG ACTTCC TTGACAACGAGGAGAACGAGGACATCCTGGAA GATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAG ATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTC GACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTA CACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGG TATTCCGGATAAACAGAGCGGTAAAACTATCCTGGATTT CCTCAAATCGGATGGCTTC GCTAATCGTAACTTCATGCA GTTGATCCACGACGACAGCCTGACCTTTAAGGAGGACAT CCAGAAAGCACAAGTGAGCGGACAGGGAGACTCACTCC ATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTA AGAAGGGAATCCTGCAAACTGTGAAGGTGGTGGACGAG CTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATAT CGTGATTGAAATGGCCCGAGAAAACCAGACTA CCCAGA AGGGCCAGAAGAACTCCCGCGAAAGGATGAAGCGGATC GAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAA AGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGA AGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGT ACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATT ACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGG ATG ACTCCATCGATAACAAGGTGTTGACTCGCAGCGACA AGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAG GTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCT GAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCT CACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATA AGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGG CAGATTACCAAGCACGTG GCGCAGATCTGGACTCCCGC ATGAACACTAAATACGACGAGAACGATAAGCTCATCCG GGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGT CGGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAG

- 150 047139

AAATCAACAACTACCATCACGCGCATGACGCATACCTCA ACGCTGTGGTCGGCACCGCCCTGATCAAGAAGTACCCTA AACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCT ACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAA ATCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAAC ATCATGAACTTCTTCAAGACTGAAATTACGCTGGCCAAT GGAGAAATCAGGAAG AGGCCACTGATCGAAACTAACGG AGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACT TCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCA ATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTT TCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAA GCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGT ACGGAGGATTCGATTCGCCGACTGTCG CATACTCCGTCC TCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAGAAG CTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATG GAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTG GAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGAT CATCAAACTGCCCAAGTACTCACTGTTCGAACTGGAAAA TGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACT CCA GAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCA ACTTCCTCTATCTTGCTTCGCACTACGAGAAACTCAAAG GGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTG GAGCAGCACAAGCATTATCTGGATGAAATCATCGAACA AATCTCCGAGTTTTTCAAAGCGCGTGATCCTCGCCGACGC CAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAG CCGATCAGAGAACAGGCCGAGAACATTATCC ACTTGTTCACCCTGACTAACCTGGGAGCTCCAGCCGCCT TCAAGTACTTCGATACTACTATCGACCGCAAAAGATACA CGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACC AAAGCATCACTGGACTCTACGAAACTAGGATCGATCTGT CGCAGCTGGGTGGCGATGGTGGCGGTGGATCCTACCCAT ACGACGTGCCTGACT ACGCCTCCGGAGGTGGTGGCCCCA AGAAGAAACGGAAGGTGTGATAG Transcript Cas9 with 5'-UTR GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGT GTGTGTGTCGTTGCAGGCCTTATTCGGATCTGCCACCAT 46

- 151 047139

HSD, OPC, corresponding to SEQ ID NO: 45, Kozak sequence and Z'-NTO ALB GGATAAGAAGTACTCGATCGGGCTGGATATCGGAACTA ATTCCGTGGGTTGGGCAGTGATCACGGATGAATACAAA GTGCCGTCCAAGAAGTTCAAGGTCCTGGGGAACACCGA TAGACACAGCATCAAGAAGAATCTCATCGGAGCCCTGCT GTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCA AACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAAT CGCATCTGCTA TCTGCAAGAAATCTTTTCGAACGAAATG GCAAAGGTGGACACAGCTTCTTCCACCGCCTGGAAGA ATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGC ATCCTATCTTTGGAAACATCGTGGACGAAGTGGCGTACC ACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAG TTGGTTGACTCAACTGACAAGGCCGACCTCAGATTGATC TACTTGGCCCTCGCCCATATGATCAAA TTCCGCGGACAC TTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGAC GTGGATAAGCTGTTCATTCAACTGGTGCAGACCTACAAC CAACTGTTCGAAGAAAACCCAATCAATGCCAGCGGCGT CGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTC GCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAG AGAAGAAGAACGGACTTTTCGGCAACTTGATCGCTCTCT CACT GGGACTCACTCCCAATTTCAAGTCCAATTTTGACC TGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACC TACGACGACGACTTGGACAATTTGCTGGCACAAATTGGC GATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTT TCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAAC ACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATT CGACGAGCATCACCAGGATCTCACGCTGCTC AAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAA GGAGATTTTCTTCGACCAGTCCAAGAATGGGTACGCAGG GTACATCGATGGAGGCGCCAGCCAGGAAGAGTTCTATA AGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACC GAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCT CCGCAAACAGAGAACCTTTGA CAACGGAAGCATTCCAC ACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGC GCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGG AAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATT

- 152 047139

ACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGT GGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGG AATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACA ATCCTTCATCGAACGAATGACCAACTTCGACAAGAATCT CCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTA CGAATACTTCACTGTCTACAACGAACTGACTAAAGTGAA ATACGTTACTGA AGGAATGAGGAAGCCGGCCTTTCTGAG CGGAGAACAGAAGAAAGCGATTGTCGATCTGCTGTTCA AGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAG GACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAA ATCAGCGGAGTGGAGGACAGATTCAACGCTTCGCTGGG AACCTATCATGATCTCCTGAAGATCATCAAGGACAAGGA CTTCCTTGACAACGAGGAGAACG AGGACATCCTGGAAG ATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGA TGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCG ACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTAC ACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGT ATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTC CTCAAATCGGATGGCTTCGCTAATCGTAACTTCAT GCAG TTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATC CAGAAAGCACAAGTGAGCGGACAGGGAGACTCACTCCA TGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAA GAAGGGAATCCTGCAAACTGTGAAGGTGGTGGACGAGC TGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATC GTGATTGAAATGGCCCGAGAAAACCAGACTACCCAGAA GCC AGAAGAACTCCCGCGAAAGGATGAAGCGGATCG AAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAA GAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAA GCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTA CGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTA CGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGA TGACTCCATCGATAAC AAGGTGTTGACTCGCAGCGACAA GAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGG TCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTG AATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTC

- 153 047139

ACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAA GGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGC AGATTACCAAGCACGTGGCGCAGATCCTGGACTCCCGCA TGAACACTAAATACGACGAGAACGATAAGCTCATCCGG GAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTC GGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGA AATCAACAACTAC CATCACGCGCATGACGCATACCTCAA CGCTGTGGTCGGCACCGCCCTGATCAAGAAGTACCCTAA ACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTA CGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAA TCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACA TCATGAACTTCTTCAAGACTGAAATTACGCTGGCCAATG GAGAAATCAGGAAGAGGCCACTGATCGA AACTAACGGA GAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTT CGCAACTGTTCGCAAAGTGCTCTTAGCCGCAAGTCAA TATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTC AAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGC TCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTAC GGAGGATTCGATTCGCCGACTGTCGCATACTCCGTCC TC GTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAGAAGCT CAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGA ACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTGGA GGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCA TCAAACTGCCCAAGTACTCACTGTTCGAACTGGAAAATG GTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAG CTGGCCTTGCCTAGCAAGTACGTCAA CTTCCTCTATCTTGCTTCGCACTACGAGAAACTCAAAGG GTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGG AGCAGCACAAGCATTATCTGGATGAAATCATCGAACAA ATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCC AACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGA GATAAGCCGATCAGAGAACAGG CCGAGAACATTATCCA CTTGTTCACCCTGACTAACCTGGGAGCTCCAGCCGCCTT CAAGTACTTCGATACTACTATCGACCGCAAAAGATACAC GTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCA

- 154 047139

AAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTC GCAGCTGGGTGGCGATGGTGGCGGTGGATCCTACCCATA CGACGTGCCTGACTACGCCTCCGGAGGTGGTGGCCCCAA GAAGAAACGGAAGGTGTGATAGCTAGCCATCACATTTA AAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAA ATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGT TGGTGTAAAGC CAACACCCTGTCTAAAAAACATAAATTT CTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATA AAAAATGGAAAGAACCTCGAG Cas9 transcript with 5'-UTR HSD, ORF corresponding to SEQ ID NO: 45, and 3'-UTR ALB GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGT GTGTGTGTCGTTGCAGGCCTTATTCGGATCTATGGATAA GAAGTACTCGATCGGGCTGGATATCGGAACTAATTCCGT GGGTTGGGCAGTGATCACGGATGAATACAAAGTGCCGT CCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACAC AGCATCAAGAAGAATCTCATCGGAGCCCTGCTGTTTG A.C. CATCTGCGGAAGAAGTTGGTTGA CTCAACTGACAAGGCCGACCTCAGATTGATCTACTTGGC CCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGAT CGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATA AGCTGTTCATTCAACTGGTGCAGACCTACAACCAACTGT TCGAAGAAAACCCAATCAATGCCAGCGGCGTCGATGCC AAGGCCATCCTGTCCGCCCGGCTGTCGAAGTC GCGGCGC CTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAGAA GAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGG ACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGA GGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACG ACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGT ACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACG TGCTGTCCGATATCCTGCGCGTGAACACCGAAA 47

- 155 047139

TAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAGCGGT ACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCG CTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGAT TTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACAT CGATGGAGGCGCCAGCCAGGAAGAGTTCTATAAGTTCA TCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAA CTGCTGGTCA AGCTGAACAGGGAGGATCTGCTCCGCAA ACAGAGAACCTTTGACAACGGAAGCATTCCACACCAGA TCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGG AGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAG ATCGAGAAAATTCTGACGTTCCGCATCCCGTATTACGTG GGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATG ACTAGAAAATCAGAGGAAACCATCACT CCTTGGAATTTC GAGGAAGTTGTGGATAAGGGAGCTTCGGCACAATCCTTC ATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAA CGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATA CTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGT TACTGAAGGAATGAGGAAGCCGGCCTTTCTGAGCGGAG AACAGAAGAAAGCGATTGTCGATCTGCTGTTCAAGA CC AACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTA CTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAG CGGAGTGGAGGACAGATTCAACGCTTCGCTGGGAACCT ATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCC TTGACAACGAGGAGAACGAGGACATCCTGGAAGATATC GTCCTGACCTTGACCCTTTTCGAGGATCGCGATGATC GAGGAGA GGCTTAAGACCTACGCTCATCTCTTCGACGAT AAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGG TTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCG CGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAA ATCGGATGGCTTCGCTAATCGTAACTTCATGCAGTTGAT CCACGACGACAGCCTGACCTTTAAGGAGGACATCCAGA AAGCACAAGTGAGCGGA CAGGGAGACTCACTCCATGAA CACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGAAG GGAATCCTGCAAACTGTGAAGGTGGTGGACGAGCTGGT GAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGA

- 156 047139

TTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGC CAGAAGAACTCCCGCGAAAGGATGAAGCGGATCGAAGA AGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGC ACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTC TACCTGTACTATTTGCAAAATGGACGGGACATGTACGTG GACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGAC GTGGACCACATCGTT CCACAGTCCTTTCTGAAGGATGAC TCCATCGATAACAAGGTGTTGACTCGCAGCGACAAGAA CAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCG TGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAAT GCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACT AAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGC TGGATTCATCAAACGGCAGCTGGTCGA GACTCGGCAGAT TACCAAGCACGTGGCGCAGATCCTGGACTCCCGCATGAA CACTAAATACGACGAGAACGATAAGCTCATCCGGGAAG TGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACT TTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCA ACAACTACCATCACGCGCATGACGCATACCTCAACGCTG TGGTCGGCACCGCCCTGATCAAGAAGTACCCTAAACTTG AATC GGAGTTTGTGTACGGAGACTACAAGGTCTACGACG TGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGG AAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATG AACTTCTTCAAGACTGAAATTACGCTGGCCAATGGAGAA ATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAAC GGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAA CTGTTCGCAAAGTGCTCTCT ATGCCGCAAGTCAATATTG TGAAGAAACCGAAGTGCAAACCGGCGGATTTTCAAAG GAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCAT TGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAG GATTCGATTCGCCGACTGTCGCATACTCCGTCCTCGTGG TGGCCAAGGTGGAGAAGGGAAAGAGCAAGAAGCTCAA ATCCGTCAAAGAGCTGCTGGGGATTACCAT CATGGAACG ATCCTCGTTCGAGAAGAACCCGATTGATTTCCTGGAGGC GAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCA AACTGCCCAAGTACTCACTGTTCGAACTGGAAAATGGTC

- 157 047139

GGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAGAAA GGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTC CTCTATCTTGCTTCGCACTACGAGAAACTCAAAGGGTCA CCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCA GCACAAGCATTATCTGGATGAAATCATCGAACAAATCTC CGAGTTTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCT CGACAAA GTCCTGTCGGCCTACAATAAGCATAGAGATA AGCCGATCAGAACAGGCCGAGAACATTATCCACTTG TTCACCCTGACTAACCTGGGAGCTCCAGCCGCCTTCAAG TACTTCGATACTACTATCGACCGCAAAAGATACACGTCC ACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAG CATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCA GCTGGGTGGCGATGGTGGCGGTG GATCCTACCCATACGA CGTGCCTGACTACGCCTCCGGAGGTGGTGGCCCCAAGAA GAAACGGAAGGTGTGATAGCTAGCCATCACATTTAAAA GCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATG AAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGG TGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTT TAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAA AAATGGAAAGAACCTCGAG Cas9 transcript containing a Cas9 ORF using m codons with typically high expression in humans GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGT GTGTGTGTCGTTGCAGGCCTTATTCGGATCCATGCCTAA GAAAAAGCGGAAGGTCGACGGGGATAAGAAGTACTCAA TCGGGCTGGATATCGGAACTAATTCCGTGGGTTGGGCAG TGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTC AAGGTCCTGGGGAACACCGATAGACACAGCATCAAGAA AAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAAC CGCAGAAGCGACCCGGCTCAAACGTACCGCGAGGCGAC GCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAG AGATCTTTTCGAACGAAATGGCAAAGGTCGACGACAGC TTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAG GACAAGAAGCATGAACGGCATCCTATCTTTGAAACATC GTCGACGAAGTGGCG TACCACGAAAAGTACCCGACCAT CTACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAA GGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATAT 48

- 158 047139

GATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATCT GAACCCTGATAACTCCGACGTGGATAAGCTTTTCATTCA ACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACC CAATCAATGCTAGCGGCGTCGATGCCAAGGCCATCCTGT CCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGA TCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTTTC GGCAACTT GATCGCTCTCTCACTGGGACTCACTCCCAAT TTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTG CAACTCTCAAAGGACACCTACGACGACGACTTGGACAA TTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTGTT CCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTC CGATATCCTGCGCGTGAACACCGAAATAACCAAAGCGC CGCTTAGCGCCTCGAT GATTAAGCGGTACGACGAGCATC ACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGC AACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGT CCAAGAATGGGTACGCAGGGTACATCGATGGAGGCGCT AGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTG GAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCT GAACAGGGAGGATCTGCTCC AAACAGAGAACCTTTG ACAACGGATCCATTCCCCACCAGATCCATCTGGGTGAGC TGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCAT TCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTG ACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGC GGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGA GGAAACCATCACTCCTTGGAATTTCGAGGAAGTT GTGGA TAAGGGAGCTTCGGCACAAAGCTTCATCGAACGAATGA CCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTC CTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACA ACGAACTGACTAAAGTGAAATACGTTACTGAAGGAATG AGGAAGCCGGCCTTTCTGTCCGGAGAACAGAAGAAAGC AATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGAC CGTCA AGCAGCTTAAAGAGGACTACTTCAAGAAGATCG AGTGTTTCGACTCAGTGGAAATCAGCGGGGTGGAGGAC AGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTG AAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGA

- 159 047139

GAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGAC CCTTTTCGAGGATCGCGATGATCGAGGAGAGGCTTAA GACCTACGCTCATCTCTTCGACGATAAGGTCATGAAACA ACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTC CCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCG GTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCG CTAATCGTA ACTTCATGCAATTGATCCACGACGACAGCC TGACCTTTAAGGAGGACATCCAAAAGCACAAGTGTCC GGACAGGGAGACTCACTCCATGAACACATCGCGAATCT GGCCGGTTCGCCGGCGATTAAGAAGGGAATTCTGCAAA CTGTGAAGGTGGTCGACGAGCTGGTGAAGGTCATGGGA CGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCG AGAAACCAGACTACCCAG AAGGGCCAGAAAAACTCCC GCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGA GCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAA ACACGCAGCTGCAGAACGAAGCTCTACCTGTACTATT TGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTG GACATCAATCGGTTGTCTGATTACGTGGACCACATC GTTCCACAGTCCTTTCTGAAGGATGACTCGAT T CGCAGATCTTGGACTCCCGCATGAACACTAAATACG ACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATT ACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGAC TTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCAT CACGCGCATGACGCATACCTCAACGCTGTGGTCGGTACC GCCCTGATCAAAAAGTACCCTAAACTTGAATCGGAGTTT GTGTACGGAGACTACAAGGTCTAC GACGTGAGGAAGAT GATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTG CGAAATACTTCTTTTACTCAAACATCATGAACTTTTTCAA GACTGAAATTACGCTGGCCAATGGAGAAATCAGGAAGA

- 160 047139

GGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATC GTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAA AGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAAC CGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGATCCT CCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGA AAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCG CCGACTGTC GCATACTCCGTCCTCGTGGTGGCCAAGGTG GAGAAGGGAAAGAGCAAAAAGCTCAAATCCGTCAAAGA GCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGA GAAGAACCCGATTGATTTCCTCGAGGCGAAGGGTTACA AGGAGGTGAAGAAGGATCTGATCATCAAACTCCCCAAG TACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATG CTGGCTTCGGCCGGAGAACT CCAAAAAGGAAATGAGCT GGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCT TCGCACTACGAAAAACTCAAAGGGTCACCGGAAGATAA CGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATT ATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAA AGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCC TGTCGGCCTACAATAAGCATAGAGATAAGCC GATCAGA GAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACT AACCTGGGAGCCCCAGCCGCCTTCAAGTACTTCGATACT ACTATCGATCGCAAAAGATACACGTCCACCAAGGAAGT TCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACT CTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGA TTGATAGTCTAGCCATCACATTTAAAAGCATCTCAGCCT ACCATGAGA ATAAGAGAAAGAAAATGAAGATCAATAGC TTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAAC ACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCC TCTTTTCTCTGTGCTTCAATTAATAAAAAAAATGGAAAGAA CCTCGAG Transcript Cas9 containing the Kozak sequence with GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGT GTGTGTGTCGTTGCAGGCCTTATTCGGATCCGCCACCAT GCCTAAGAAAAAGCGGAAGGTCGACGGGGATAAGAAGT ACTCAATCGGGCTGGATATCGGAACTAATTCCGTGGGTT GGGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAG 49

- 161 047139

Cas9 ORF using m codons with generally high expression in humans AAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCAT CAAGAAAAATCTCATCGGAGCCCTGCTGTTTGACTCCGG CGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGCGA GGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATC TGCAAGAGATCTTTTCGAACGAAATGGCAAAGGTCGAC GACAGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTG GAGGAGGACAA GAAGCATGAACGGCATCCTATCTTTGG AAACATCGTCGACGAAGTGGCGTACCACGAAAAGTACC CGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTCAA CTGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCG CCCATATGATCAAATTCCGCGGACACTTCCTGATCGAAG GCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTT TCATTCAACTGGTGCAGACCTACAACCA ACTGTTCGAAG AAAACCCAATCAATGCTAGCGGCGTCGATGCCAAGGCC ATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAA AACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGG ACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACT CCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACCGG AAGCTGCAACTCTCAAGGACACCTACGACGACGACTT GGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGG ATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTT GCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAA AGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGA GCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAG ACAGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGA CAAGAATGGGTACGCAGGGTACATCGATGGAG GCGCTAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAA TCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTC AAGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAAC CTTTGACAACGGATCCATTCCCCACAGATCCATCTGGG TGAGCTGCACGCCATCTTGCGGCGCCAGGGAGGACTTTTA CCCATTCCTCAAGGACAACCGG GAAAAGATCGAGAAAA TTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGG CGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAAT CAGAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTG

- 162 047139

TGGATAAGGGAGCTTCGGCACAAAGCTTCATCGAACGA ATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGT GCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTC TACAACGAACTGACTAAAGTGAAATACGTTACTGAAGG AATGAGGAAGCCGGCCTTTCTGTCCGGAGAACAGAAGA AAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGG AAGCAGCTTAAAGAGGACTACTTCAAGAAG ATCGAGTGTTTCGACTCAGTGGAAATCAGCGGGGTGGA GGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCT CCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACG AGGAAACGAGGACATCCTGGAAGATATCGTCCTGACC TTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGG CTTAAGACCTACGCTCATCTCTTC GACGATAAGGTCATG AAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCG CCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACA GAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATGG CTTCGCTAATCGTAACTTCATGCAATTGATCCACGACGA CAGCCTGACCTTTAAGGAGGACATCCAAAAAGCACAAG TGTCCGGACAGGGAGACTCACTCCATGAACACAT CGCG AATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATTCTG CAAACTGTGAAGGTGGTCGACGAGCTGGTGAAGGTCAT GGGACGGCACAAACCGGAGAATATCGTGATTGAAATGG CCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAAAAC TCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAA GGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGG AAAACACGCA GCTGCAGAACGAGAAGCTCTACCTGTAC TATTTGCAAAATGGACGGGACATGTACGTGGACCAAGA GCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCA CATCGTTCCACAGTCCTTTCTGAAGGATGACTCGATCGA TAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGA AGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAG ATGAAGAATTACTG GCGGCAGCTCCTGAATGCGAAGCT GATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCGA GCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCAT CAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGC

- 163 047139

ACGTGGCGCAGATCTTGGACTCCCGCATGAACACTAAAT ACGACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTG ATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAG GACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTAC CATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGT ACCGCCCTGATCAAAAAGTACCCTAAACTTGAATCGGAG TTTGTGTACGGAGACTACAA GGTCTACGACGTGAGGAA GATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAA CTGCGAAATACTTCTTTTACTCAAACATCATGAACTTTTT CAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGA AGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAA ATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGC AAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAG AAA ACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGAT CCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCA AGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGAT TCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAG GTGGAGAAGGGAAAGAGCAAAGCTCAAATCCGTCAA AGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTT CGAGAAGAACCC GATTGATTTCCTCGAGGCGAAGGGTT ACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCCCC AAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCG CATGCTGGCTTCGGCCGGAGAACTCCAAAAGGAAATG AGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATC TTGCTTCGCACTACGAAAAACTCAAAGGGTCACCGGAA GATAACGAACAGAAGCAGCTTTTCGTG GAGCAGCACAA GCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTT TTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAA AGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGAT CAGAGAACAGGCCGAGAACATTATCCACTTGTTCACCCT GACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACTTCGA TACTACTATCGATCGCAAAAGATACACGTCCACCAAGGA AGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGG ACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGG CGATTGATAGTCTAGCCATCACATTTAAAAGCATCTCAG

- 164 047139

CCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAT AGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCC AACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTT GCCTCTTTTCTCTGTGCTTCAATTAATAAAAAAAATGGAAA GAACCTCGAG Cas9 ORFs with deleted splice points; U content 12.75% ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAAC AAACAGCGTCGGATGGGCAGTCATCACAGACGAATACA AGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACA GACAGACACAGCATCAAGAAGAACCTGATCGGAGCACT GCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGAC TGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAA GAACAGAATCT GCTACCTGCAGGAAATCTTCAGCAACG AAATGGCAAAGGTCGACGACAGCTTCTTCCACcggCTGG AAGAAAGCTTCCTGGTCGAAGAAGACAAAGAAGCACGAA AGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGC ATACCACGAAAAGTACCCGACAATCTACCACCTGAGAA AGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGA CTGATCTACCTGGCACTGGCACACAT GATCAAGTTCAGA GGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAA CAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGAC ATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAA GCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTG AGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCT GCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCT GA TCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGC AACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAG CAAGGACACATACGACGACGACCTGGACAACCTGCTGG CACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCA GCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACAT CCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGA GCGCAAGCATGATCAA GAGATACGACGAACACCACCAG GACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCT GCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCA AGAACGGATACGCAGGATACATCGACGGAGGAGCAAGC CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAA 50

- 165 047139

AAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAA CAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACA ACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTG CACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTC CTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGAC ATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGG AAACAGCAGAT TCGCATGGATGACAAGAAAGAGCGAAG AAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGAC AAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGAC AAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGC CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACA ACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATG AGAAAGCCGGCATTCCTGAGCGGA GAACAGAAGAAGGC AATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCA CAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATC GAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGA CAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCT GAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAG AAAACGAAGACATCCTGGAAGACATCGTCCTGACACT G ACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACT GAAGACATACGCACACCTGTTCGACGACAAGGTCATGA AGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAG ACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGC AGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGAC GGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGAC GACAGCCTGACAT TCAAGGAAGACATCCAGAAGGCACA GGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCG CAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATC CTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGT CATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAA TGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAA GAACAGCAGAGAAAGAATGAAGAGAATCGA AGAAGGA ATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCC GGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACC TGTACTACCTGCAaAACGGAAGAGACATGTACGTCGACC

- 166 047139

AGGAACTGGACATCAACAGACTGAGCGACTACGACGTC GACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAG CATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACA GAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTC AAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGC AAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAA AGGCAGAGAGA GGAGGACTGAGCGAACTGGACAAGGC AGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGA TCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATG AACACAAAGTACGACGAAAACGACAAGCTGATCAGAGA AGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCG ACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAA ATCAACAACTACCACCACGCACACGA CGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGA AGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTC TACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGA AATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCA ACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAAC AAACAGGAGAAATCGTCTGGGACAAGGGAAGAG ACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAG GTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGG ATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCG ACAAGCTGATCGCAAGAAAGAAGGACTGGGACCGAAG AAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAG CGTCCTGGTCGTCGCAAAGG TCGAAAAGGGAAAGAGCA AGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACA ATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGA CTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAAC TGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAG CA AGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAA AGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCA GCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAAT

- 167 047139

CATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCC TGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATAC AACAAGCACAGACAAGCCGATCAGAGAACAGGCAG AAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCA ACACTGATCCACC AGAGCATCACAGGACTGTACGAAAC AAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAG GAAGCCCGAAGAAGAAGAGAAAGGTCTAG Cas9 transcript with 5' UTR HSD, ORF corresponding to SEQ ID NO: 50, Kozak sequence and Z'-NTO ALB GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGT GTGTGTGTCGTTGCAGGCCTTATTCGGATCCGCCACCAT GGACAAGAAGTACAGCATCGGACTGGACATCGGAACAA ACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAG GTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGA CAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGC GACAGCGGAGAAACAGCAGAAGCAACAAGACTG AAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGA ACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAA ATGGCAAAGGTCGACGACAGCTTCTTCCACcggCTGGAA GAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAG ACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATA CCACGAAAAGTACCCGACAAT CTACCACCTGAGAAAGA AGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTG ATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGA CACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAG CGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATA CAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCG GAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGA GC AAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCC GGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCG CACTGAGCCTGGACTGACACCGAACTTCAAGAGCAAC TTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAA GGACACATACGACGACGACCTGGACAACCTGCTGGCAC AGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCA AAGAACCTGAGC GACGCAATCCTGCTGAGCGACATCCT 51

- 168 047139

GAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCG CAAGCATGATCAAGAGATACGACGAACACCACCAGGAC CTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCC GGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGA ACGGATACGCAGGATACATCGACGAGGAGCAAGCCAG GAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAA GAACTGCTGGTCAAGCTGAACA GAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAAC GGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCA CGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCT GAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACAT TCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGA AACAGCAGATTCGCATGGATGACAAGAAAGAGCGA AGA AACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACA AGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACA AACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCC GAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAA CGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGA GAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCA ATCGTCGA CCTGCTGTTCAAGACAAACAGAAAGGTCAC AGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCG AATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGAC AGATTCAACGCAAGCCTGGGAACATACCACGACCTGCT GAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAG AAAACGAAGACATCCTGGAAGACATCGTCCTGACACTG ACACTGTTCGAAGACAGAGAAAT GATCGAAGAAAGACT GAAGACATACGCACACCTGTTCGACGACAAGGTCATGA AGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAG ACTGAGCAGAAAGCTGATCAACGGAATCAGACAAGC AGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGAC GGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGAC GACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACA GGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCG CAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATC CTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGT

- 169 047139

CATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAA TGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAA GAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGA ATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCC GGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACC TGTACTACCTGCAaAACGGAAGAGACATGTACGTCGACC AGGAACTGGACAT CAACAGACTGAGCGACTACGACGTC GACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAG CATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACA GAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTC AAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGC AAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAA AGGCAGAGAGAGGACTGAGCGAACT GGACAAGGC AGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGA TCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATG AACACAAAGTACGACGAAAACGACAAGCTGATCAGAGA AGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCG ACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAA ATCAACAACTACCACCACGCACACGACGCATACCTGAA CGC AGTCGTCGGAACAGCACTGATCAAGAAGTACCCGA AGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTC TACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGA AATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCA ACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAAC GGAGAAACAGGAGAAATCGTC TGGGACAAGGGAAGAG ACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAG GTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGG ATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCG ACAAGCTGATCGCAAGAAAGAAGGACTGGGAACCCGAAG AAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAG CGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAG CA AGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACA ATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGA CTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG

- 170 047139

ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAAC TGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCA AGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAA AGCTGAAGGAAGCCCGGAAGACAACGAACAGAAGCA GCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAAT CATCGAAC AGATCAGCGAATTCAGCAAGAGAGTCATCC TGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATAC AACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAG AAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCA ACACTGATCCACCAGAGCATCACA A ATTAATAAAAATGGAAAGAACCTCGAG OPC Cas9 with minimal uridine content codons, often used in humans in general; U content 12.75% ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCAC CAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACA AGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACC GACAGACACAGCATCAAGAAGAACCTGATCGGCGCCCT GCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCAGAC TGAAGAGAACCGCCAGAAGAAGATACACCAGAAGAAA GAACAGAATCTGC TACCTGCAGGAGATCTTCAGCAACG AGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTG GAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGA GAGACACCCCATCTTCGGCAACATCGTGGACGAGGTGG CCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAA AGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGAGA CTGATCTACCTGGCCCTGGCCCA CATGATCAAGTTCAGA GGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAAC AGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACC 52

- 171 047139

TACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAG CGGCGTGGACGCCAAGGCCATCCTGAGCGCCAGACTGA GCAAGAGCAGAAGACTGGAGAACCTGATCGCCCAGCTG CCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGAT CGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAA CTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCA AGGACACCTAC GACGACGACCTGGACAACCTGCTGGCC CAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCC AAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTG AGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGC CAGCATGATCAAGATACGACGAGCACCACCAGGACC TGACCCTGCTGAAGGCCCTGGTGAGACAGCAGCTGCCCG AGAAGTACAAGGAGATCTTCTTC GACCAGAGCAAGAAC GGCTACGCCGGCTACATCGACGCGGCGCCAGCCAGGA GGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGAT GGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACAGAG AGGACCTGCTGAGAAAGCAGAGAACCTTCGACAACGGC AGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCC ATCCTGAGAAGACAGGAGGACTTCTACCCCTTC CTGAAG GACAACAGAGAGAAGATCGAAGATCCTGACCTTCAG AATCCCCTACTACGTGGGCCCCCTGGCCAGAGGCAACAG CAGATTCGCCTGGATGACCAGAAAGAGCGAGGAGACCA TCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGC GCCAGCGCCCAGAGCTTCATCGAGAGAATGACCAACTTC GACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCA CA GCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCT GACCAAGGTGAAGTACGTGACCGAGGGCATGAGAAAGC CCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG GACCTGCTGTTCAAGACCAACAGAAAGGTGACCGTGAA GCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCT TCGACAGCGTGGAGATCAGCGGCGTGGAGGACAGATGG AACGCCAGCCTGC ACCTACCACGACCTGCTGAAGATC ATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGA GGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTT CGAGGACAGAGATGATCGAGGAGAGACTGAAGACCT

- 172 047139

ACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTG AAGAGAAGAAGATACACCGGCTGGGGCAGACTGAGCAG AAAGCTGATCAACGGCATCAGAGACAAGCAGAGCGGCA AGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCA ACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTG ACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGG CCAGGGCGACAGCC TGCACGAGCACATCGCCAACCTGG CCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACC GTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCAG ACACAAGCCCGAGAACATCGTGATCGAGATGGCCAGAG AGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCAG AGAGAATGAAGAGAATCGAGGAGGGCATCAAGGAG CTGGGCAGCCAGATCCTGAAGGAGCACCCCG TGGAGAA CACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCT GCAGAACGGCAGAGACATGTACGTGGACCAGGAGCTGG ACATCAACAGACTGAGCGACTACGACGTGGACCACATC GTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAA CAAGGTGCTGACCAGAAGCGACAAGAACAGAGGCAAGA GCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATG AAGAACTACTGGAGACAGCTGCTGAACGCCAAGCTGAT CACCCAGAGAAAGTTCGACAACCTGACCAAGGCCGAGA GAGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATC AAGAGACAGCTGGTGGAGACCAGACAGATCACCAAGCA CGTGGCCCAGATCCTGGACAGCAGAATGAACACCAAGT ACGACGAGAACGACAAGCTGATCAGAGAGGTGAAGGTG AGCAAGCTGGTGAGCGACTTCAGAAA GGACTTCCAGTTCTACAAGGTGAGAGAGATCAACAACT ACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGG GCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGC GAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGAG AAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGG CCACCGCCAAGTACTTCTTCTAC AGCAACATCATGAACT TCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCA GAAAGAGACCCCTGATCGAGACCAACGGCGAGACCGGC GAGATCGTGTGGGACAAGGGCAGAGACTTCGCCACCGT

- 173 047139

GAGAAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGA AGAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAG AGCATCCTGCCCAAGAGAAACAGCGACAAGCTGATCGC CAGAAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCT TCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGG CCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAGAG CGTGAAGGAG CTGCTGGGCATCACCATCATGGAGAGAA GCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCA AGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAG CTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCAG AAAGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAGG GCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCC TGTACCTGGCCAGCCACTACGA GAAGCTGAAGGGCAGC CCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCA GCACAAGCACTACCTGGACGAGATCATCGAGCAGATCA GCGAGTTCAGCAAGAGAGTGATCCTGGCCGACGCCAAC CTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGA CAAGCCCATCAGAGAGCAGGCCGAGAACATCATCCACC TGTTCACCCTGACCAACCTGGGCGCCCCCGCCG CCTTCA AGTACTTCGACACCACCATCGACAGAAAGAGATACACC AGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCA GAGCATCACCGGCCTGTACGAGACCAGAATCGACCTGA GCCAGCTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAG AAGAGAAAGGTGTGA Cas9 transcript with 5'-UTR HSD, ORF corresponding to SEQ ID NO: 52, Kozak sequence and Z'-NTO ALB GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGT GTGTGTGTCGTTGCAGGCCTTATTCGGATCCGCCACCAT GGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCA ACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAG GTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGA CAGACACAGCATCAAGAAGAACCTGATCGGCGCCCTGC ACAGCGGCGAGACCGCCGAGGCCACCAGACTG AAGAGAACCGCCAGAAGAAGATACACCAGAAGAAAGA ACAGAATCTGCTACCTGCAGGAGATCTTCAGCAACGAG ATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGA GGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGA 53

- 174 047139

GACACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCT ACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAG AAGCTGGTGGACAGCACCGACAAGGCCGACCTGAGACT GATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGG CCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAG CGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTA CAACCAGCTGTTCG AGGAGAACCCCATCAACGCCAGCG GCGTGGACGCCAAGGCCATCCTGAGCGCCAGACTGAGC AAGAGCAGAAGACTGGAGAACCTGATCGCCCAGCTGCC CGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCG CCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACT TCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAG GACACCTACGACGACGACCTGGACAACCT GCTGGCCCA GATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAA GAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGAG AGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCA GCATGATCAAGAGATACGACGAGCACCACCAGGACCTG ACCCTGCTGAAGGCCCTGGTGAGACAGCAGCTGCCCGA GAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACG GCT ACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAG GAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATG GACGGCACCGAGGAGCTGCTGGTGAAGCTGAACAGAGA GGACCTGCTGAGAAAGCAGAGAACCTTCGACAACGGCA GCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCA TCCTGAGAAGACAGGAGGACTTCTACCCCTTCCTGAAGG ACAACAGAGAGAAG ATCGAGAAGATCCTGACCTTCAGA ATCCCCTACTACGTGGGCCCCCTGGCCAGAGGCAACAGC AGATTCGCCTGGATGACCAGAAAGAGCGAGGAGACCAT CACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCG CCAGCGCCCAGAGCTTCATCGAGAGAATGACCAACTTCG ACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCAC AGCCTGCTGTACGAGTACTTCACCGTGTA CAACGAGCTG ACCAAGGTGAAGTACGTGACCGAGGGCATGAGAAAGCC CGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGG ACCTGCTGTTCAAGACCAACAGAAAGGTGACCGTGAAG

- 175 047139

CAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTT CGACAGCGTGGAGATCAGCGGCGTGGAGGACAGATTCA ACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCA TCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAG GACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTC GAGGACAGAGAGATGATCGAGGAGAGACTGAAGACCTA CG CCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGA AGAGAAGAAGATACACCGGCTGGGGCAGACTGAGCAGA AAGCTGATCAACGGCATCAGAGACAAGCAGAGCGGCAA GACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAA CAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGAC CTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCC AGGGCGACA GCCTGCACGAGCACATCGCCAACCTGGCC GGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGT GAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCAGAC ACAAGCCCGAGAACATCGTGATCGAGATGGCCAGAGAG AACCAGACCACCCAGAAGGGCCAGAAGAACAGCAGAG AGAGAATGAAGAGAATCGAGGAGGGCATCAAGGAGCTG GGCAGCCAGATCCTGAAGGAGC ACCCCGTGGAGAACAC CCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCA GAACGGCAGAGACATGTACGTGGACCAGGAGCTGGACA TCAACAGACTGAGCGACTACGACGTGGACCACATCGTG CCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAA GGTGCTGACCAGAAGCGACAAGAACAGAGGCAAGAGCG ACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGAT GAAG AACTACTGGAGACAGCTGCTGAACGCCAAGCTGATCAC CCAGAGAAAGTTCGACAACCTGACCAAGGCCGAGAGAG GCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAG AGACAGCTGGTGGAGACCAGACAGATCACCAAGCACGT GGCCCAGATCCTGGACAGCAGAATGAACACCAAGTACG ACGAGAACGACAAGCTGATCAGAGAGGTGAAGGTGATC AGCAAGCTGGTGAGCGACTTCAGAAAGGA CTTCCAGTTCTACAAGGTGAGAGAGATCAACAACTACCA CCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCAC CGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGT

- 176 047139

TCGTGTACGGCGACTACAAGGTGTACGACGTGAGAAAG ATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCAC CGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTT CAAGACCGAGATCACCCTGGCCAACGGCGAGATCAGAA AGAGACCCCTGATCGAGACCAACGGCGAGACCGGCGAG ATCGTGTGGGACAAGGGCAGAGACTTCGCCACCGTGAG AAAGGTGCTGAG CATGCCCCAGGTGAACATCGTGAAGA AGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGC ATCCTGCCCAAGAGAAACAGCGACAAGCTGATCGCCAG AAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCG ACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCA AGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGT GAAGGAGCTGCTGGGCATCACCATCAT GGAGAGAAGCA GCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAG GGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCT GCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCAGAA AGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGC AACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTG TACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCC CGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGC ACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGC GAGTTCAGCAAGAGAGTGATCCTGGCCGACGCCAACCT GGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACA AGCCCATCAGAGAGCAGGCCGAGAACATCATCCACCTG TTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAG TACTTCGACACC ACCATCGACAGAAAGAGATACACCAG CACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGA GCATCACCGGCCTGTACGAGACCAGAATCGACCTGAGC CAGCTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAA GAGAAAGGTGTGACTAGCCATCACATTTAAAAGCATCTC AGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCA ATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGT AAAG CCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCAT TTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGA AAGAACCTCGAG

- 177 047139

Cas9 ORF with minimal uridine content codons, rarely used in humans in general; U content 12.75% ATGGACAAAAAATACAGCATAGGGCTAGACATAGGGAC GAACAGCGTAGGGTGGGCGGTAATAACGGACGAATACA AAGTACCGAGCAAAAAATTCAAAGTACTAGGGAACACG GACCGACACAGCATAAAAAAAAACCTAATAGGGGCGCT ACTATTCGACAGCGGGGAAACGGCGGAAGCGACGCGAC TAAAACGAACGGCGCGACGACGATACACGCGACGAAAA AACCGAATATGCTACCTACAAGAAA TATTCAGCAACGA AATGGCGAAAGTAGACGACAGCTTCTTCCACCGACTAG AAGAAAGCTTCCTAGTAGAAGAAGACAAAAAACACGAA CGACACCCGATATTCGGGAACATAGTAGACGAAGTAGC GTACCACGAAAAATACCGCGATATACCACCTACGAA AAAAACTAGTAGACAGCACGGACAAAGCGGACCTACGA CTAATATACCTAGCGCTAGCGCACATGATAAAATTCCGA TCCTAATAGAAGGGGACCTAAACCCGGACAA CAGCGACGTAGACAAACTATTCATACAACTAGTACAAA CGTACAACCAACTATTCGAAGAAAACCCGATAAACGCG AGCGGGGTAGACGCGAAAGCGATACTAAGCGCGCGACT AAGCAAAAGCCGACGACTAGAAAACCTAATAGCGCAAC TACCGGGGGAAAAAAAAAACGGGCTATTCGGGAACCTA ATAGCGCTAAGCCTAGGGCTAACGCCGAACT TCAAAAG CAACTTCGACCTAGCGGAAGACGCGAAACTACAACTAA GCAAAGACACGTACGACGACGACCTAGACAACCTACTA GCGCAAATAGGGGACCAATACGCGGACCTATTCCTAGC GGCGAAAAACCTAAGCGACGCGATACTACTAAGCGACA TACTACGAGTAAACACGGAAATAACGAAAGCGCCGCTA AGCCGAGCATGATAAAACGATACGACGAACACCACA AGACCTAACGCT ACTAAAAGCGCTAGTACGACAACAAC TACCGGAAAAATACAAAGAAATATTCTTCGACCAAAGC AAAAACGGGTACGCGGGGTACATAGACGGGGGGGCGAG CAAGAAGAATTCTACAAATTCATAAAACCGATACTAG AAAAAATGGACGGGACGGAAGAACTAGTAAAACTA AACCGAGAAGACCTACTACGAAAACAACGAACGTTCGA CAACGGGAGCATACCGCACCAAATACACCTAGGGGAAC TACACGCGATACTACGACGACAAGAAGACTTCTACCCGT TCCTAAAAGACAACCGAGAAAAAATAGAAAAAATACTA 54

- 178 047139

ACGTTCCGAATACCGTACTACGTAGGGCCGCTAGCGCGA GGGAACAGCCGATTCGCGTGGATGACGCGAAAAAGCGA AGAAACGATAACGCCGTGGAACTTCGAAGAAGTAGTAG ACAAAGGGGCGAGCGCGCAAAGCTTCATAGAACGAATG ACGAACTTCGACAAAACCTACCGAACGAAAAAGTACT ACCGAAACACAGCCTACTATACGAATACTTCACGGTATA CAACGAACTAACGAAAG TAAAATACGTAACGGAAGGGA TGCGAAAACCGGCGTTCCTAAGCGGGGAACAAAAAAAA GCGATAGTAGACCTACTATTCAAAACGAACCGAAAAGT AACGGTAAAACAACTAAAAGAAGACTACTTCAAAAAAA TAGAATGCTTCGACAGCGTAGAAATAAGCGGGGTAGAA GACCGATTCAACGCGAGCCTAGGGACGTACCACGACCT ACTAAAAATAATAAAAGACAAAGACTTCCTAGACAACG AAG AAAACGAAGACATACTAGAAGACATAGTACTAACG CTAACGCTATTCGAAGACCGAGAAATGATAGAAGAACG ACTAAAAACGTACGCGCACCTATTCGACGACAAAGTAA TGAAACAACTAAAACGACGACGATACACGGGGTGGGGG CGACTAAGCCGAAAACTAATAAACGGGATACGAGACAA ACAAAGCGGGAAAACGATACTAGACTTCCTAAAAAGCG ACGGGTTCGCGAACCGAAACTTCATGCA ACTAATACACG ACGACAGCCTAACGTTCAAAGAAGACATACAAAAAGCG CAAGTAAGCGGGCAAGGGGACAGCCTACACGAACACAT AGCGAACCTAGCGGGGAGCCCGGCGATAAAAAAAGGGA TACTACAAACGGTAAAAGTAGTAGACGAACTAGTAAAA GTAATGGGGCGACACAAACCGGAAAACATAGTAATAGA AATGGCGCGAGAAAACCAAACGACGCAAAAGGGCAA AAAACAGCCGAGAACGA ATGAAACGAATAGAAGAAG GGATAAAAGAACTAGGGAGCCAAATACTAAAAGAACAC CCGGTAGAAAACACGCAACTACAAAACGAAAAACTATA CCTATACTACCTACAAAACGGGCGAGACATGTACGTAG ACCAAGAACTAGACATAAACCGACTAAGCGACTACGAC GTAGACCACATAGTACCGCAAAGCTTCCTAAAAGACGA CAGCATAGACAACAAAGTACTAACGCGAAGCGACAAAA ACCGAGGGAAAAGCGA CAACGTACCGAGCGAAGAAGTA GTAAAAAAAATGAAAAACTACTGGCGACAACTACTAAA

- 179 047139

CGCGAAACTAATAACGCAACGAAAATTCGACAACCTAA CGAAAGCGGAACGAGGGGGGCTAAGCGAACTAGACAA AGCGGGGTTCATAAAACGACAACTAGTAGAAACGCGAC AAATAACGAAACACGTAGCGCAAATACTAGACAGCCGA ATGAACACGAAATACGACGAAAACGACAAACTAATACG AGAAGTAAAAGTAATAACGCTAAAAAGCAAACTAGTAA GCGACTTCCGAAAAGACTTCCAAT TCTACAAAGTACGAG AAATAAACAACTACCACCACGCGCACGACGCGTACCTA AACGCGGTAGTAGGGACGGCGCTAATAAAAAAATACCC GAAACTAGAAAGCGAATTCGTATACGGGGACTACAAAG TATACGACGTACGAAAAATGATAGCGAAAAGCGAACAA GAAATAGGGAAAGCGACGGCGAAATACTTCTTCTACAG CAACATAATGAACTTCTTCAAAACGGAAATAACGCTAGC GGAAATACGAAAACGACCGCTAATAGAAACGA ACGGGGAAACGGGGGAAATAGTATGGGACAAAGGGCG AGACTTCGCGACGGTACGAAAAGTACTAAGCATGCCGC AAGTAAACATAGTAAAAAAAACGGAAGTACAAACGGGG GGGTTCAGCAAAGAAAGCATACTACCGAAACGAAACAG CGACAAACTAATAGCGCGAAAAAAAGACTGGGAACCCGA AAAAATACGGGGGTTCGACAGCCCGAC TAGCGTAC AGCGTACTAGTAGTAGCGAAAGTAGAAAAAGGGAAAAG CAAAAAACTAAAAAGCGTAAAAGAACTAGGGATAA CGATAATGGAACGAAGCAGCTTCGAAAAAAACCCGATA GACTTCCTAGAAGCGAAAGGGTACAAAGAAGTAAAAAA AGACCTAATAATAAAACTACCGAAATACAGCCTATTCGA ACTAGAAAACGGGCGAAAACGAATGCTAGCGAGCGCGG GGGAACTACAAAAAGGGA ACGAACTAGCGCTACCGAGC AAATACGTAAACTTCCTATACCTAGCGAGCCACTACGAA AAACTAAAAGGGAGCCCGGAAGACAACGAACAAAAAC AACTATTCGTAGAACAACACAAACACTACCTAGACGAA ATAATAGAACAAATAAGCGAATTCAGCAAACGAGTAAT ACTAGCGGACGCGAACCTAGACAAAGTACTAAGCGCGT ACAACAAACACCGAGACAAACCGATACGAGAACAAG CG GAAAACATAATACACCTATTCACGCTAACGAACCTAGG GGCGCCGGCGGCGTTCAAATACTTCGACACGACGATAG

- 180 047139

ACCGAAAACGATACACGAGCACGAAAGAAGTACTAGAC GCGACGCTAATACACCAAAGCATAACGGGGCTATACGA AACGCGAATAGACCTAAGCCAACTAGGGGGGGACGGGG GGGGGAGCCCGAAAAAAAAACGAAAAGTATGA Cas9 transcript with 5' UTR HSD, ORF corresponding to SEQ ID NO: 54, Kozak sequence and Z'-NTO ALB GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGT GTGTGTGTCGTTGCAGGCCTTATTCGGATCCGCCACCAT GGACAAAAAATACAGCATAGGGCTAGACATAGGGACGA ACAGCGTAGGGTGGGCGGTAATAACGGACGAATACAAA GTACCGAGCAAAAAATTCAAAGTACTAGGGAACACGGA CCGACACAGCATAAAAAAAAACCTAATAGGGGCGCTAC TATTCGACAGCGGGGAAA CGGCGGAAGCGACGCGACTA AAACGAACGGCGCGACGACGATACACGCGACGAAAAAA CCGAATATGCTACCTACAAGAAATATTCAGCAACGAAAT GGCGAAAGTAGACGACAGCTTCTTCCACCGACTAGAAG AAAGCTTCCTAGTAGAAGAAGACAAAAAACACGAACGA CACCCGATATTCGGGAACATAGTAGACGAAGTAGCGTA CCACGAAAAATACCCCGACGATATACCACCTACGAAAA A AACTAGTAGACAGCACGGACAAAGCGGACCTACGACTA ATATACCTAGCGCTAGCGCACATGATAAAATTCCGAGGG CACTTCCTAATAGAAGGGGACCTAAACCCGGACAACAG CGACGTAGACAAACTATTCATACAACTAGTACAAACGTA CAACCAACTATTCGAAGAAAACCCGATAAACGCGAGCG GGGTAGACGCGAAAGCGATACTAAGCGCGCGACTAAGC AAAAGCCGACGACTAGAAAA CCTAATAGCGCAACTACC GGGGGAAAAAAAAAACGGGCTATTCGGGAACCTAATAG CGCTAAGCCTAGGGCTAACGCCGAACTTCAAAAGCAAC TTCGACCTAGCGGAAGACGCGAAACTACAACTAAGCAA AGACACGTACGACGACCTAGACAACCTACTAGCGC AAATAGGGGACCAATACGCGGACCTATTCCTAGCGGCG AAAACCTAAGCGACGCGATACTACTAAGCGACATACT ACGAG TAAACACGGAAATAACGAAAGCGCCGCTAAGCG CGAGCATGATAAAACGATACGACGAACACCACCAAGAC CTAACGCTACTAAAAGCGCTAGTACGACAACAACTACC GGAAAAATACAAAGAAATATTCTTCGACCAAAGCAAAA ACGGGTACGCGGGGTACATAGACGGGGGGGCGAGCCAA 55

- 181 047139

GAAGAATCTACAAATTCATAAAACCGATACTAGAAAA AATGGACGGGACGGAAGAACTAGTAAAACTAAACC GAGAAGACCTACGAAAACAACGAACGTTCGACAAC GGGAGCATACCGCACCAAATACACCTAGGGGAACTACA CGCGATACTACGACGACAAGAAGACTTCTACCCGTTCCT AAAAGACAACCGAGAAAAAATAGAAAAATACTAACGT TCCGAATACCGTACTACG TAGGGCCGCTAGCGCGAGGG AACAGCCGATTCGCGTGGATGACGCGAAAAAGCGAAGA AACGATAACGCCGTGGAACTTCGAAGAAGTAGTAGACA AAGGGGCGAGCGCGCAAAGCTTCATAGAACGAATGACG AACTTCGACAAAACCTACCGAACGAAAAAGTACTACC GAAACACAGCCTACTATACGAATACTTCACGGTATACAA CGAACTAACGAAAGTAAAATACGTAACGGAAGGG GC GAAAACCGGCGTTCCTAAGCGGGGAACAAAAAAAAGCG ATAGTAGACCTACTATTCAAAACGAACCGAAAAGTAAC GGTAAAACAACTAAAAGAAGACTTCAAAAAAATAG AATGCTTCGACAGCGTAGAAATAAGCGGGGTAGAAGAC CGATTCAACGCGAGCCTAGGGACGTACCACGACCTACTA AAAATAATAAAAGACAAAGACTTCCTAGACAACGAAGA AAACGAAGACATACTAGAAGA CATAGTACTAACGCTAA CGCTATTCGAAGACCGAGAAATGATAGAAGAACGACTA AAAACGTACGCGCACCTATTCGACGACAAAGTAATGAA ACAACTAAAACGACGATACACGGGGTGGGGGCGAC TAAGCCGAAAACTAATAAACGGGATACGAGACAAACAA AGCGGGAAAACGATACTAGACTTCCTAAAAAGCGACGG GTTCGCGAACCGAAACTTCATGCAACTAATACACGACGA AACGTTCAAAGAAGACATACAAAAAGCGCAAG TAAGCGGGCAAGGGGACAGCCTACACGAACACATAGCG AACCTAGCGGGGAGCCCGGCGATAAAAAAAGGGATACT ACAAACGGTAAAAGTAGTAGACGAACTAGTAAAAGTAA TGGGGCGACACAAACCGGAAAACATAGTAATAGAAATG GCGCGAGAAAACCAAACGACGCAAAAAGGGCAAAAAA ACAGCCGAGAACGAATGAAACGAATAGAAGAAG GGAT AAAAGAACTAGGGAGCCAAATACTAAAAGAACACCCGG TAGAAAACACGCAACTACAAAACGAAAAACTATACCTA

- 182 047139

TACTACCTACAAAACGGGCGAGACATGTACGTAGACCA AGAACTAGACATAAACCGACTAAGCGACTACGACGTAG ACCACATAGTACCGCAAAGCTTCCTAAAAGACGACAGC ATAGACAACAAAGTACTAACGCGAAGCGACAAAACCG AGGGAAAAGCGACAACGTACCGAGCGAAGAAGTAGTAA AAAAAATGAAAAACTACTGGCGACAACTACTAAACGCG AAACTAATAACGCAACGAAA ATTCGACAACCTAACGAA AGCGGAACGAGGGGGGCTAAGCGAACTAGACAAAGCG GGGTTCATAAAACGACAACTAGTAGAAACGCGACAAAT AACGAAACACGTAGCGCAAATACTAGACAGCCGAATGA ACACGAAATACGACGAAAACGACAAACTAATACGAGAA GTAAAAGTAATAACGCTAAAAAGCAAACTAGTAAGCGA CTTCCGAAAAGACTTCCAATTCTACAAAGTACGAGAAAT AAACA ACTACCACCACGCGCACGACGCGTACCTAAACG CGGTAGTAGGGACGGCGCTAATAAAAAAATACCCGAAA CTAGAAAGCGAATTCGTATACGGGGACTACAAAGTATA CGACGTACGAAAAATGATAGCGAAAAGCGAACAAGAAA TAGGGAAAGCGACGGCGAAATACTTCTTCTACAGCAAC ATAATGAACTTCTTCAAAACGGAAATAACGCTAGCGAA CGGGGAAATACGAAAACGACCGCTAATAG AAACGAACG GGGAAACGGGGGAAATAGTATGGGACAAAGGGCGAGA CTTCGCGACGGTACGAAAAGTACTAAGCATGCCGCAAG TAAACATAGTAAAAAAAACGGAAGTACAAACGGGGGGG TTCAGCAAAGAAAGCATACTACCGAAACGAAACAGCGA CAAACTAATAGCGCGAAAAAAAGACTGGGACCGGAAAA AATACGGGGGTTCGACAGCCCGACGGTAGCGTACAGC GTACTAGTAGTAGCGAAA GTAGAAAAAGGGAAAAGCAA AAAACTAAAAAGCGTAAAAGAACTACTAGGGATAACGA TAATGGAACGAAGCAGCTTCGAAAAAAACCCGATAGAC TTCCTAGAAGCGAAAGGGTACAAAGAAGTAAAAAAAGA CCTAATAATAAAACTACCGAAATACAGCCTATTCGAACT AGAAACGGGCGAAAACGAATGCTAGCGAGCGCGGGG GAACTACAAAAAGGGAACGAACTAGCGCTACCGAGCAA ATACGTAA ACTTCCTATACCTAGCGAGCCACTACGAAAA ACTAAAAGGGAGCCCGGAAGACAACGAACAAAAACAA

- 183 047139

CTATTCGTAGAACAACACAAACACTACCTAGACGAAAT AATAGAACAAATAAGCGAATTCAGCAAACGAGTAATAC TAGCGGACGCGAACCTAGACAAAGTACTAAGCGCGTAC AACAAACACCGAGACAAACCGATACGAGAACAAGCGGA AAACATAATACACCTATCACGCTAACGAACCTAGGGGC GCCGGCGGCGTTCAAATACTTCGACACGACGATAGACC GAAAACGATA CACGAGCACGAAAGAAGTACTAGACGCG ACGCTAATACACCAAAGCATAACGGGGCTATACGAAAC GCGAATAGACCTAAGCCAACTAGGGGGGGACGGGGGGG GGAGCCCGAAAAAAAAACGAAAAGTATGACTAGCCATC ACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAG AAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTTC TTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAA AAC ATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTC AATTAATAAAAAAAATGGAAAGAACCTCGAG Cas9 transcript with AGG as the first three nucleotides FOR use with CleanCap™, 5'-UTR HSD, ORF, corresponding to SEQ ID NO: 4, sequence Kozak and Z’-NTO ALB AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGT GTGTGTGTCGTTGCAGGCCTTATTCGGATCCGCCACCAT GGACAAGAAGTACAGCATCGGACTGGACATCGGAACAA ACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAG GTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGA CAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGC GACAGCGGAGAAACAGCAGAAGCAACAAGACTG AAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGA ACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAA ATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGA AGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAA GACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCAT ACCACGAAAAGTACCCCGACAAT CTACCACCTGAGAAAG AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACT GATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGG ACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACA GCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACAT ACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGC GGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGA G CAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGC 56

- 184 047139

CGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATC GCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAA CTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCA AGGACACATACGACGACGACCTGGACAACCTGCTGGCA CAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGC AAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCC TGAGAGTCAACA CAGAAATCACAAAGGCACCGCTGAGC GCAAGCATGATCAAGAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCC GGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGA ACGGATACGCAGGATACATCGACGGAGCAAGCCAG GAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAACTGCTGGTCAA GCTGAACA GAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAAC GGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCA CGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCT GAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACAT TCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGA AACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGA ACCGTGGAACTTCGAAGAAGTCGTCGACA AGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACA AACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCC GAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAA CGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGA GAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCA ATCGTCGACCTGCTGTTCAAG ACAAACAGAAAGGTCAC AGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCG AATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGAC AGATTCAACGCAAGCCTGGGAACATACCACGACCTGCT GAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAG AAAACGAAGACATCCTGGAAGACATCGTCCTGACACTG ACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACT GAAGACATACGCACACCTGTTCGACGACAAGGTCATGA AGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAG ACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGC

- 185 047139

AGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGAC GGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGAC GACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACA GGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCG CAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATC CTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGT CATGGGAAG ACACAAGCCGGAAAACATCGTCATCGAAA TGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAA GAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGA ATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCC GGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACC TGTACTACCTGCAGAACGGAAGAGACATGTACGTCGAC CAGGAACTGGACATCAACAGACTGAGCG CAG GATTCATCAAGAGACAGCTGGTCGAAACAAGACAG ATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAAT GAACACAAAGTACGACGAAAACGACAAGCTGATCAGAG AAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGC GACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGA AATCAACAACTACCACCACGCACACGACGCATACCTGA ACGCAGTCGTCGGAACA GCACTGATCAAGAAGTACCCG AAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGT CTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGG AAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGC AACATCATGAACTTCTTCAAGACAGAAATCACACTGGCA AACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAA CGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAA GA GACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCA GGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAG GATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGC

- 186 047139

GACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACA GCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGC AAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCAC AATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCG ACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAG GACCTGATCATCAAG CTGCCGAAGTACAGCCTGTTCGAA CTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCA AGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAA AGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCA GCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAAT CATCGAACAGATCAGCGAATTCAG CAAGAGAGTCATCC TGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATAC AACAAGCACAGACAAGCCGATCAGAGAACAGGCAG AAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCA ACACTGATCCACCAGAGCATCACAGGACTGTACGAAAC A AGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAG GAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATC ACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAG AAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTC TTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAC ATAAATTTCTTTAATCATTTTGCCTCTTTTCTTGTGCTTC AATTAATAAAAAATGGAAA GAACCTCGAG Cas9 transcript with 5'-UTR from CMV, ORF corresponding to SEQ ID NO: 4, Kozak sequence and Z'-NTO ALB GGGCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGAC CTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGG CCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCA AGAGTGACTCACCGTCCTTGACACGGCCACCATGGACAA GAAGTACAGCATCGGACTGGACATCGGAACAAACAGCG TCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCG AGCAAGA AGTTCAAGGTCCTGGGAAACACAGACAGACA CAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCG ACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAG 57

- 187 047139

AACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGA ATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCA AAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAG CTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACC CGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACG AAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTG GTCGACAGCA CAGACAAGGCAGACCTGAGACTGATCTA CCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTT CCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACG TCGAAAGCTGTTCATCCAGCTGGTCCAGACATACAACC AGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTC GACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAG CAGAAGACTGGAAAACCTGATCGCACAGCTGC CGGGAG AAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGA CCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACA CATACGACGACGACCTGGACAACCTGCTGGCACAGATC GGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAA CCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGT AAATCACAAAGGCACCGCTGAGCGCAAGCA TGATCAAGAGATACGACGAACACCACCAGGACCTGACA CTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAA GTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGAT ACGCAGGATACATCGACGGAGCAAGCCAGGAAGAA TTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGAC GGAACAGAAGAACTGCTGGTCAAGCT GAACAGAGAAGA CCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCA TCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCC TGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAAT CCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCA GATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATC ACAC CGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGC AAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCG ACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCAC

- 188 047139

AGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTG ACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCG ACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAG CAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTT CGACACGTCGAAATCAGCGGAGTCGAAGACAGATTCA ACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATC ATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGA AGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACAT ACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTG AAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCA GAAAGCTGATCAACGGAATCAGAGAC AAGCAGAGCGGA AAGACAATCTGGACTTCCTGAAGAGCGACGGATTCGC AAACAGAAACTTCATGCAGCTGATCCACGACGACAGCC TGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGC GGACAGGGAGACAGCCTGCACGAACACATCGCAAACCT GGCAGGAAGCCCGCAATCAAGAAGGGAATCCTGCAGA CAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGA AGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAG AGAAAACCAGACAACACAGAAGGGACAGAAGAACAGC AGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGG AACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAA AACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTA CCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAAC TGGACATCAACAGACT GAGCGACTACGACGTCGACCAC ATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGAC AACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAA AGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAG ATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCT GATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAG AGAGAGGAGGACTGAGCGAACTGGACAAGG CAGGATTC ATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAA GCACGTCGCACAGATCCTGGACAGCAGAATGAACACAA AGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAG

- 189 047139

GTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAG AAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACA ACTACCACCACGCACACGACGCATACCTGAACGCAGTC GTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGA AAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGT CAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAA AGGCAACAGCAAAG TACTTCTTCTACAGCAACATCATGA ACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAA ATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAAC AGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAA CAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATC GTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAA GGAAAGCATCCTGCCGAAGAGAAACAGCGACAAG CTGA TCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGA GGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTC GTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGA AGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAA AGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGA AGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCA TCAAGCTGCCGA AD CCTGGCAGACG CAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCAC AGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCAT CCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGC ATTCAAGTACTTCGACACAACAATCGACAGAAAGAGAT ACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATC CACCAGAGCATCACAGGACTGTACGAAACAAGAATCGA CCTGAGCC AGCTGGGAGGAGACGGAGGAGGAAGCCCGA AGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAA AGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAAT

- 190 047139

GAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTG GTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCT TTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAA AAAATGGAAAGAACCTCGAG Cas9 transcript with 5'-UTR from HBB, ORF corresponding to SEQ ID NO: 4, Kozak sequence and Z'-NTO NVV GGGacatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccggatctgcc accATGGACAAGAAGTACAGCATCGGACTGGACATCGGA ACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATA CAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACA CAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCA CTGCTGTTCGACAGCGG AGAAACAGCAGAAGCAACAAG ACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGA AAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAA CGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACT GGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACG AAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTC GCATACCACGAAAAGTACCCCGACAATCTACCACCTGAG AAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGA GACTGATCTACCTGGCACTGGCACACATGATCAAGTTCA GAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGAC AACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAG ACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGC AAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGAC CAGAAGACTGGAAAACCTGATCGCACAG CTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCT GATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGA GCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTG AGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGG CAGCAAAGAACCTGAGCGACGCAATCCTG CTGAGCGAC ATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCT GAGCGCAAGCATGATCAAGAGATACGACGAACACCACC AGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAG CTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAG CAAGAACGGATACGCAGGATACATCGACGGAGGAGCAA GCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGG 58

- 191 047139

AAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTG AACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGA CAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAAC TGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGT TCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTG ACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGA GGAAACAGCAG ATTCGCATGGATGACAAGAAAGAGCGA AGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCG ACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATG ACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCT GCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTA CAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAA TGAGAAAGCCGGCATTCCTGAGCGGA C TGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAG ACTGAAGACATACGCACACCTGTTCGACGACAAGGTCAT GAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGA AGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAA GCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCG ACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACG ACGACAGCCTGACATTCAA GGAAGACATCCAGAAGGCA CAGGTCAGCGGACAGGGACAGCCTGCACGAACACAT CGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAA TCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAG GTCATGGGAAGACAAGCCGGAAAACATCGTCATCGA AATGGCAAGAGAAAACCAGACACACAGAAGGGACAG AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAG GAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTA CCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCG

- 192 047139

ACCAGGAACTGGACATCAACAGACTGAGCGACTACGAC GTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGAC AGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAA CAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCG TCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAAC GCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGAC AAAGGCAGAGAGA GGAGGACTGAGCGAACTGGACAAG GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACA GATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAA TGAACACAAAGTACGACGAAAACGACAAGCTGATCAGA GAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAG CGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGA AATCAACACTACCACCACGCACAC GACGCATACCTGA ACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCG AAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGT CTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGG AAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGC AACATCATGAACTTCTTCAAGACAGAAATCACACTGGCA AACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAA AAACAGGAGAAATCGTCTGGGACAAGGGAAGA GACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCA GGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAG GATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGC GACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACA GCGTCCTGGTCGTCGCAAAGG TCGAAAAGGGAAAGAGC AAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCAC AATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCG ACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAG GACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAA CTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCA AGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAA AGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCA GCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAAT

- 193 047139

CATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCC TGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATAC AACAAGCACAGACAAGCCGATCAGAGAACAGGCAG AAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCA ACACTGATCCACC AGAGCATCACAGGACTGTACGAAAC AAGAATCGACCTGAGCCAGCTGGGAGGACGGAGGAG GAAGCCCGAAGAAGAAGAGAAAGGTCTAGctagcgctcgctttct tgctgtccaatttctattaaaggttcctttgttccctaagtccaactaaactgggggatattatg aagggccttgagcatctggattctgcctaataaaaacatttatt ttcattgcctcgag Cas9 transcript with 5'-UTR from XBG, ORF corresponding to SEQ ID NO: 4, sequence Kozak and Z’-NTO XBG GGGaagctcagaataaacgctcaactttggccggatctgccacCATGGACAAGA AGTACAGCATCGACTGGACATCGGAACAAACAGCGTC GGATGGGCAGTCATCACAGACGAATACAAGGTCCCGAG CAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACA GCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGAC AGCGGAGAAACAGCAGAAGCAACAAGACT GAAGAGAA CAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAAT CTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAA AGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGC TTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCC GATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGA AAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGG TCGACA GCACAGACAAGGCAGACCTGAGACTGATCTAC CTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTC CTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGT CGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCA GCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCG ACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGC AGAAGACTGGAAAACCTGATCGC ACAGCTGCGGGAGA AAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGA GCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACC TGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA TACGACGACGACCTGGACAACCTGCTGGCACAGATCGG AGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACC 59

- 194 047139

TGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCA ACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATG ATCAAGAGATACGACGAACACCACCAGGACCTGACACT GCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGT ACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATAC GCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATT CTACAAGTTCATCAAGCCGA TCCTGGAAAAGATGGACG GAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGAC CTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCAT CCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCT GAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACA ACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATC CCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGC AG ATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCA CACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGA CAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACA GCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGA CAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCG GCATTCCTGAGCAG AACAGAAGAAGGCAATCGTCGA CCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGC AGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAA CGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCA TCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAA GACATCCTGGAACATCGTCCTGACACTGAC ACTGTTC GAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATA CGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGA AGAGAAGAAGATACACAGGATGGGAAGACTGAGCAG AAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCA AACAGAAACTTCATGCAGCTGATCCACGACGACAGCCT GA CATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTG GCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGAC

- 195 047139

AGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAA GACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCA GAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGA ACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAA ACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTAC CTGCAGAACGGAA GAGACATGTACGTCGACCAGGAACT GGACATCAACAGACTGAGCGACTACGACGTCGACCACA TCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACA ACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAA GAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGA TGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTG ATCACACAGAGAAAGTTCGACAACCTGAC AAAGGCAGA GAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCA TCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAG CACGTCGCACAGATCCTGGACAGCAGAATGAACACAAA GTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGG TCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGA AAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAA CCACGCACACGACGCATACCTGAACGCAGTCGT CGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAA GCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCA GAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAA GGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAA CTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAA TCAGAAAGAGACCGCTGAT CGAAACAAACGGAGAAACA GGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAAC AGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAG GAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGAT CGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGA GGATTCGACAGCCCGACAGTCGCATACAGCGTCCT GGTC GTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGA AGACGTCAAGGAACTGCTGGGAATCACAATCATGGAA AGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGA

- 196 047139

AGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCA TCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAAC GGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCA GAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCA ACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGG GAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTC GAACAGCACAA GCACTACCTGGACGAAATCATCGAACA GATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACG CAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCAC AGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCAT CCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGC ATTCAAGTACTTCGACACAACAATCGACAGAAAGAGAT ACACAAGCACAAAGGAAGTCCTGGACG CAACACTGATC CACCAGAGCATCACAGGACTGTACGAAACAAGAATCGA CCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGA AGAAGAAGAGAAAGGTCTAGctagcaccagcctcaagaacacccgaat ggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccat tcgtatctgctcctaataaaagaaagtt tcttcacattctctcgag Cas9 transcript with AGG as the first three nucleotides FOR use with CleanCap™, 5'-UTO from XBG, ORF corresponding to SEQ ID NO: 4, sequence Kozak and Z’-NTO XBG AGGaagctcagaataaacgctcaactttggccggatctgccacCATGGACAAGA AGTACAGCATCGACTGGACATCGGAACAAACAGCGTC GGATGGGCAGTCATCACAGACGAATACAAGGTCCCGAG CAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACA GCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGAC AGCGGAGAAACAGCAGAAGCAACAAGACT GAAGAGAA CAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAAT CTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAA AGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGC TTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCC GATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGA AAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGG TCGACA GCACAGACAAGGCAGACCTGAGACTGATCTAC CTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTC CTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGT CGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCA GCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCG 60

- 197 047139

ACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGC AGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGA AAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGA GCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACC TGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA TACGACGACGACCTGGACAACCTGCTGGCACAGATCGG AGACCAGTACGCAGA CCTGTTCCTGGCAGCAAAGAACC TGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCA ACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATG ATCAAGAGATACGACGAACACCACCAGGACCTGACACT GCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGT ACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATAC GCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAA TT CTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACG GAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGAC CTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCAT CCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCT GAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACA ACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATC CCGTACTACGTCGG ACCGCTGGCAAGAGGAAACAGCAG ATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCA CACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGA CAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACA GCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGA CAAAGGTCAAGTACGTCACAGAAGGAATGAG AAAGCCG GCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGA CCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGC AGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAA CGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCA TCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAA AGACATCGTCCTGACACTGACACTGTTC GAAGACAGAAAATGATCGAAGAAAGACTGAAGACATA CGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGA

- 198 047139

AGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAG AAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCA AACAGAAACTTCATGCAGCTGATCCACGACGACAGCCT GACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTG GCAGGAAGCCCG GCAATCAAGAAGGGAATCCTGCAGAC AGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAA GACAAAGCCGGAAAACATCGTCATCGAAATGGCAAGA GAAACCAGACAACACAGAAGGGACAGAAGAACAGCA GAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGA ACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAA ACACACAGCTGCAGAACGAAAAGCTGTA CCTGTACTAC CTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACT GGACATCAACAGACTGAGCGACTACGACGTCGACCACA TCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACA ACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAA GAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGA TGAAGAACTACTGGACAGCTGCTGAACGCAAAGCTG ATCAC ACAGAGAAAGTTCGACAACCTGACAAAGGCAGA GAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCA TCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAG CACGTCGCACAGATCCTGGACAGCAGAATGAACACAAA GTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGG TCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGA AAGGACTTCCAGTTCTACAA TCAGAGAAATCAACAA CTACCACCACGCACACGACGCATACCTGAACGCAGTCGT CGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAA GCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCA GAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAA GGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAA CTTCTTCAAGACAGAAATCACACTGGCAAACGGA GAAA TCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACA GGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAAC AGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG

- 199 047139

TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAG GAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGAT CGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGA GGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTC GTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGA AGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAA AGAAGCAGCTTCG AAAAGAACCCGATCGACTTCCTGGA AGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCA TCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAAC GGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCA GAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCA ACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGG GAAGCCCGGAAGACAACGAACAGAAGCA GCTGTTCGTC GAACAGCACAAGCACTACCTGGACGAAATCATCGAACA GATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACG CAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCAC AGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCAT CCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGC ATTCAAGTACTTCGACACAACAATCGACAGAAAGAGAT GCACAAAGGAAGTCCTGGACGCAACACTGATC CACCAGAGCATCACAGGACTGTACGAAACAAGAATCGA CCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGA AGAAGAAGAGAAAGGTCTAGctagcaccagcctcaagaacacccgaat ggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccat tcgtatctgct cctaataaaaagaaagtttcttcacattctctcgag Cas9 transcript with AGG as the first three nucleotides FOR use with CleanCap™, 5'-UTO from AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGT GTGTGTGTCGTTGCAGGCCTTATTCGGATCCGCCACCAT GGACAAGAAGTACAGCATCGGACTGGACATCGGAACAA ACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAG GTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGA CAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGC GACAGCGGAGAAACAGCAGAAGCAACAAGACTG AAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGA ACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAA ATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGA 61

- 200 047139

HSD, ORF corresponding to SEQ ID NO: 4, Kozak sequence and Z'-NTO ALB AGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAA GACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCAT ACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACT GATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGG ACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACA GCGACGTCGA CAAGCTGTTCATCCAGCTGGTCCAGACAT ACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGC GGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAG CAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGC CGGGAAAAGAAGAACGGACTGTTCGGAAACCTGATC GCACTGAGCCTGGACTGACACCGAACTTCAAGAGCAA CTTCGACCTGGCAGAAGACGCAAAGCTG CAGCTGAGCA AGGACACATACGACGACGACCTGGACAACCTGCTGGCA CAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGC AAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCC TGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGC GCAAGCATGATCAAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCC AGTACAAGGAAATCTTCTTCGACCAGAGCAAGA ACGGATACGCAGGATACATCGACGAGGAGCAAGCCAG GAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACA GGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAAC GGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCA CGCAATCCTGAGAAGACAGGAAG ACTTCTACCCGTTCCT GAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACAT TCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGA AACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGA AACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACA AGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACA AACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCC GAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAA CGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGA GAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCA

- 201 047139

ATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCAC AGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCG AATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGAC AGATTCAACGCAAGCCTGGGAACATACCACGACCTGCT GAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAG AAAACGAAGACATCCTGGAACATCGTCCTGACACTG ACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACT GAAGACATACGCACACCTGTTCGACGACAAGGTCATGA AGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAG ACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGC AGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGAC GGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGAC GACAGCCTGACATTCAAGGAAGACATCCAG AAGGCACA GGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCG CAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATC CTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGT CATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAA TGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAA GAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGA ACTGGGAAGCCAGATCCTGAAGGAACACCC GGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACC TGTACTACCTGCAGAACGGAAGAGACATGTACGTCGAC CAGGAACTGGACATCAACAGACTGAGCGACTACGACGT CGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACA GCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAAC AGAGGAAAGAGCGACAACG TCCCGAGCGAAGAAGTCGT CAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACG CAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACA AAGGCAGAGAGGAGGACTGAGCGAACTGGACAAGG CAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAG ATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAAT GAACACAAAGTACGACGAAAACGACAAGCTGATC AGAG AAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGC GACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGA AATCAACAACTACCACCACGCACACGACGCATACCTGA

- 202 047139

ACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCG AAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGT CTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGG AAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGC AACATCATGAACTTCTTCAAGACAGAAATCACACTGGCA AACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAA CGGAGAAACAGGAGAAA TCGTCTGGGACAAGGGAAGA GACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCA GGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAG GATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGC GACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACA GCGTCCTGGTCGTCGCAAAGGTCGAAAAGGG AAAGAGC AAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCAC AATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCG ACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAG GACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAA CTGGAAAACGGAAGAAAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCA AGTACGTC AACTTCCTGTACCTGGCAAGCCACTACGAAA AGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCA GCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAAT CATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCC TGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATAC AACAAGCACAGACAAGCCGATCAGAGAACAGGCAG AAAACATCATCCACCTGTTC ACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCA ACACTGATCCACCAGAGCATCACAGGACTGTACGAAAC AAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAG GAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATC ACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAG AAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTTC TTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAAAC ATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTC

- 203 047139

AATTAATAAAAATGGAAAGAACCTCGAG 30/30/39 poly-A sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAGCGAAAA AAAAAAAAAAAAAAAAAAAAAAAACCGAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAA 62 sequence poly-A 100 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 63 Guide RNA G209 mC*mC*mA*GUCCAGCGAGGCAAAGGGUUUUAGAGCUA GAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA CUUGAAAAAGUGGCACCGAGUCGGUGCmU*mU*mU*U 64 ORF encoding Neisseria meningitidis Cas9 ATGGCAGCATTCAAGCCGAACTCGATCAACTACATCCTG GGACTGGACATCGGAATCGCATCGGTCGGATGGGCAAT GGTCGAAATCGACGAAGAAGAAAACCCGATCAGACTGA TCGACCTGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTC CCGAAGACAGGAGACTCGCTGGCAATGGCAAGAAGACT GGCAAGATCGGTCAGAAGACTGACAAGAAGAAGAGCAC ACAGACTGCTGA GAACAAGAAGACTGCTGAAGAGAGAA GGAGTCCTGCAGGCAGCAAACTTCGACGAAAACGGACT GATCAAGTCGCTGCCGAACACACGTGGCAGCTGAGAG CAGCAGCACTGGACAGAAAGCTGACACCGCTGGAATGG TCGGCAGTCCTGCTGCACCTGATCAAGCACAGAGGATAC CTGTCGCAGAGAAAGAACGAAGGAGAAACAGCAGACA AGGAACTGGGAGCACTGCTGAAGAG TCGCAGGAAAC GCACACGCACTGCAGACAGGAGACTTCAGAACACCGGC AGAACTGGCACTGAACAAGTTCGAAAAGGAATCGGGAC ACATCAGAAACCAGAGATCGGACTACTCGCACACATTCT CGAGAAAGGACCTGCAGGCAGAACTGATCCTGCTGTTC GAAAAGCAGAAGGAATTCGGAAACCCGCACGTCTCGGG AGGACTGAAGGAAGGAATCGAAACACTGCTGATGACAC AGA GACCGGCACTGTCGGGAGACGCAGTCCAGAAGATG CTGGGACACTGCACATTCGAACCGGCAGAACCGAAGGC AGCAAAGAACACATACACAGCAGAAAGATTCATCTGGC TGACAAAGCTGAACAACCTGAGAATCCTGGAACAGGGA TCGGAAAGACCGCTGACAGACACAGAAAGAGCAACACT GATGGACGAACCGTACAGAAAGTCGAAGCTGACATACG 65

- 204 047139

CACAGGCAAGAAAGCTGCTGGGACTGGAAGACACAGCA TTCTTCAAGGGACTGAGATACGGAAAGGACAACGCAGA AGCATCGACACTGATGGAAATGAAGGCATACCACGCAA TCTCGAGAGCACTGGAAAAGGAAGGACTGAAGGACAAG AAGTCGCCGCTGAACCTGTCGCCGGAACTGCAGGACGA AATCGGAACAGCATTCTCGCTGTTCAAGACAGACGAAG ACATCACAGGAAGACT GAAGGACAGAATCCAGCCGGAA ATCCTGGAAGCACTGCTGAAGCACATCTCGTTCGACAAG TTCGTCCAGATCTCGCTGAAGGCACTGAGAAGAATCGTC CCGCTGATGGAACAGGGAAAGAGATACGACGAAGCATG CGCAGAAATCTACGGAGACCACTACGGAAAGAAGAACA CAGAAGAAAAGATCTACCTGCCGCCGATCCCGGCAGAC GAAATCAGAAACCCGTCGTCCTGAGAGCACT GTCGCA GGCAAGAAAGGTCATCAACGGAGTCGTCAGAAGATACG GATCGCCGGCAAGAATCCACATCGAAACAGCAAGAGAA GTCGGAAAGTCGTTCAAGGACAGAAAGGAAATCGAAAA GAGACAGGAAGAAAACAGAAAGGACAGAGAAAAGGCA GCAGCAAAGTTCAGAGAATACTTCCCGAACTTCGTCGGA GAACCGAAGTCGAAGGACATCCTGAAGCTGAGACTGTA CGAACAGCA GCACGGAAAGTGCCTGTACTCGGGAAAGG AAATCAACCTGGGAAGACTGAACGAAAAGGGGATACGTC GAAATCGACCACGCACTGCCGTTCTCGAGAACATGGGA CGACTCGTTCAACAACAAGGTCCTGGTCCTGGGATCGGA AAACCAGAACAAGGGAAACCAGACACCGTACGAATACT TCAACGGAAAGGACAACTCGAGAGAATGGCAGGAATTC AAGGCAAGAGTCGAAACATCGAGATTC CCGAGATCGAA GAAGCAGAAATCCTGCTGCAGAAGTTCGACGAAGACG GATTCAAGGAAAGAAACCTGAACGACACAAGATACGTC AACAGATTCCTGTGCCAGTTCGTCGCAGACAGAATGAGA CTGACAGGAAAGGGAAAGAAGAGAGTCTTCGCATCGAA CGGACAGATCACAAACCTGCTGAGAGGATTCTGGGGAC TGAGAAAGGTCAGAGCAGAAAACGACAGACACCACGCA CTGGACGCAGTCGTCGTCGCATGCTCGACAGTCGCAATG CAGCAGAAGATCACAAGATTCGTCAGATACAAGGAAAT GAACGCATTCGACGGAAAGACAATCGACAAGGAAACAG

- 205 047139

GAGAAGTCCTGCACCAGAAGACACACTTCCCGCAGCCG TGGGAATTCTTCGCACAGGAAGTCATGATCAGAGTCTTC GGAAAGCCGGACGGAAAGCCGGAATTCGAAGAAGCAG ACACACTGGAAAAGCTGAGAACACTGCTGGCAGAAAAG CTGTCGTCGAGACCGGAAGCAGTCCACGAATACGTCAC ACCGCTGTTCGTCTCGAGAGCACCGAACAGAAAGATGTC GGGACAGGGACA CATGGAAACAGTCAAGTCGGCAAAGA GACTGGACGAAGGAGTCTCGGTCCTGAGAGTCCCGCTG ACACAGCTGAAGCTGAAGGACCTGGAAAAGATGGTCAA CAGAGAAAGAGAACCGAAGCTGTACGAAGCACTGAAGG CAAGACTGGAAGCACACAAGGACGACCCGGCAAAGGCA TTCGCAGAACCGTTCTACAAGTACGACAAGGCAGGAAA CAGAACACAGCAGGTCAAGGCAGTCAGAG TCGAACAGG TCCAGAAGACAGGAGTCTGGGTCAGAAACCACAACGGA ATCGCAGACAACGCAACAATGGTCAGAGTAGACGTCTT CGAAAAGGGAGACAAGTACTACCTGGTCCCGATCTACTC GTGGCAGGTCGCAAAGGGAATCCTGCCGGACAGAGCAG TCGTCCAGGGAAAGGACGAAGAAGACTGGCAGCTGATC GACGACTCGTTCAACTTCAAGTTCTCGCTGCACCCGAAC GAC CTGGTCGAAGTCATCACAAAGAAGGCAAGAATGTT CGGATACTTCGCATCGTGCCACAGAGGAACAGGAAACA TCAACATCAGAATCCACGACCTGGACCACAAGATCGGA AAGAACGGAATCCTGGAAGGAATCGGAGTCAAGACAGC ACTGTCGTTCCAGAAGTACCAGATCGACGAACTGGGAA AGGAAATCAGACCGTGCAGACTGAAGAAGAGACCGCCG GTCAGATCCGGAAAGA GAACAGCAGACGGATCGGAATT CGAATCGCCGAAGAAGAAGAGAAAGGTCGAATGA OPC encoding Neisseria meningitidis Cas9 (no start or stop codons; suitable for inclusion in GCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGA CTGGACATCGGAATCGCATCGGTCGGATGGGCAATGGTC GAAATCGACGAAGAAGAAAACCCGATCAGACTGATCGA CCTGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTCCCGA AGACAGGAGACTCGCTGGCAATGGCAAGAAGACTGGCA AGATCGGTCAGAAGACTGACAAGAAGAAGAGCACACAG ACTGCTGAGAACAA GAAGACTGCTGAAGAGAGAAGGAG TCCTGCAGGCAGCAAACTTCGACGAAAACGGACTGATC 66

- 206 047139

sequence encoding the fusion protein) AAGTCGCTGCCGAACACACCGTGGCAGCTGAGAGCAGC AGCACTGGACAGAAAGCTGACACCGCTGGAATGGTCGG CAGTCCTGCTGCACCTGATCAAGCACAGAGGATACCTGT CGCAGAGAAAGAACGAAGGAGAAACAGCAGACAAGGA ACTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCAC ACGCACTGCAGACAGGAGACTTCAGAACACCGGCAGAA CTGGCACTGAA CAAGTTCGAAAAGGAATCGGGACACAT CAGAAACCAGAGATCGGACTACTCGCACACATTCTCGA GAAAGGACCTGCAGGCAGAACTGATCCTGCTGTTCGAA AAGCAGAAGGAATTCGGAAACCCGCACGTCTCGGGAGG ACTGAAGGAAGGAATCGAAACACTGCTGATGACACAGA GACCGGCACTGTCGGGAGACGCAGTCCAGAAGATGCTG GGACACTGCACATTCGAACCGGCAGAACCGA AGGCAGC AAAGAACACATACACAGCAGAAAGATTCATCTGGCTGA CAAAGCTGAACAACCTGAGAATCCTGGAACAGGGATCG GAAAGACCGCTGACAGACACAGAAAGAGCAACACTGAT GGACGAACCGTACAGAAAGTCGAAGCTGACATACGCAC AGGCAAGAAAGCTGCTGGGACTGGAAGACACAGCATTC TTCAAGGGACTGAGATACGAAAGGACAACGCAGAAGC ATCGACACTG ATGGAAATGAAGGCATACCACGCAATCT CGAGAGCACTGGAAAAGGAAGGACTGAAGGACAAGAA GTCGCCGCTGAACCTGTCGCCGGAACTGCAGGACGAAA TCGGAACAGCATTCTCGCTGTTCAAGACAGACGAAGAC ATCACAGGAAGACTGAAGGACAGAATCCAGCCGGAAAT CCTGGAAGCACTGCTGAAGCACATCTCGTTCGACAAGTT CGTCCAGATCTCGCTGAAGGCACTGAGAAGAA TCGTCCC GCTGATGGAACAGGGAAAGAGATACGACGAAGCATGCG CAGAAATCTACGGAGACCACTACGGAAAGAAGAACACA GAAGAAAAGATCTACCTGCCGCCGATCCCGGCAGACGA AATCAGAAACCCGGTCGTCCTGAGAGCACTGTCGCAGG CAAGAAAGGTCATCAACGGAGTCGTCAGAAGATACGGA TCGCCGGCAAGAATCCACATCGAAACAGCAAGAGAAGT CGGAAAGTC GTTCAAGGACAGAAAGGAAATCGAAAAGA GACAGGAAGAAAACAGAAAGGACAGAGAAAAGGCAGC AGCAAAGTTCAGAGAATACTTCCCGAACTTCGTCGGAGA

- 207 047139

ACCGAAGTCGAAGGACATCCTGAAGCTGAGACTGTACG AACAGCAGCACGGAAAGTGCCTGTACTCGGGAAAGGAA ATCAACCTGGGAAGACTGAACGAAAAGGGATACGTCGA AATCGACCACGCACTGCCGTTCTCGAGAACATGGGACG ACTCGTTCAACAACAAGGTCCTGGTCCTGGGATCGGAAA ACCAGAACAAGGGAAACCAGACACCGTACGAATACTTC AACGGAAAGGACAACTCGAGAG AATGGCAGGAATTCAA GGCAAGAGTCGAAACATCGAGATTCCCGAGATCGAAGA AGCAGAGAATCCTGCTGCAGAAGTTCGACGAAGACGGA TTCAAGGAAAGAAACCTGAACGACACAAGATACGTCAA CAGATTCCTGTGCCAGTTCGTCGCAGACAGAATGAGACT GACAGGAAAGGAAAGAAGAGAGTCTTCGCATCGAACG GACAGATCACAAACCTGCTGAGAGGATTCTGGACT A GCCGGACGGAAAGCCGGAATTCGAAGAAGCAGACA CACTGGAAAAGCTGAGAACACTGCTGGCAGAAAAGCTG TCGTCGAGACCGGAAGCAGTCCACGAATACGTCACACC GCTGTTCGTCTCGAGAGCACCGAACAGAAAGATGTCGG GACAGGGACACATGGAAACAGTCAAGTCGGCAAAGAGA CTGGACGAAGGAGTCTCGGTCCTGAGAGTCCCGCTGACA CAGCTGAAGCTGAA GGACCTGGAAAAGATGGTCAACAG AGAAAGAGAACCGAAGCTGTACGAAGCACTGAAGGCAA GACTGGAAGCACAAGGACGACCCGGCAAAGGCATTC GCAGAACCGTTCTACAAGTACGACAAGGCAGGAAACAG AACACAGCAGGTCAAGGCAGTCAGAGTCGAACAGGTCC AGAAGACAGGAGTCTGGGTCAGAAACCACAACGGAATC GCAGACAACGCAACAATGGTCAGAGTA GACGTCTTCGA AAAGGGAGACAAGTACTACCTGGTCCCGATCTACTCGTG GCAGGTCGCAAAGGGAATCCTGCCGGACAGAGCAGTCG TCCAGGGAAAGGACGAAGAAGACTGGCAGCTGATCGAC

- 208 047139

GACTCGTTCAACTTCAAGTTCTCGCTGCACCCGAACGAC CTGGTCGAAGTCATCACAAAGAAGGCAAGAATGTTCGG ATACTTCGCATCGTGCCACAGAGGAACAGGAAACATCA ACATCAGAATCCACGACCTGGACCACAAGATCGGAAAG AACGGAATCCTGGAAGGAATCGGAGTCAAGACAGCACT GTCGTTCCAGAAGTACCAGATCGACGAACTGGGAAAGG AAATCAGACCGTG CAGACTGAAGAAGAGACCGCCGGTC AGATCCGGAAAGAGAACAGCAGACGGATCGGAATTCGA ATCGCCGAAGAAGAAGAGAAAGGTCGAA Transcript containing SEQ ID NO: 65 (encoding Neisseria meningitidis Cas9) GGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGG ATCCGCCACCATGGCAGCATTCAAGCCGAACTCGATCAA CTACATCCTGGGACTGGACATCGGAATCGCATCGGTCGG ATGGGCAATGGTCGAAATCGACGAAGAAGAAAACCCGA TCAGACTGATCGACCTGGGAGTCAGAGTCTTCGAAAGA GCAGAAGTCCCGAAGACAGGAGACTCGCTGGCAATGGC AAGAAGACT GGCAAGATCGGTCAGAAGACTGACAAGAA GAAGAGCACACAGACTGCTGAGAACAAGAAGACTGCTG AAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGA AAACGGACTGATCAAGTCGCTGCCGAACACACGTGGC AGCTGAGAGCAGCAGCACTGGACAGAAAGCTGACACCG CTGGAATGGTCGGCAGTCCTGCTGCACCTGATCAAGCAC AGAGGATACCTGTCGCAGAG AAAGAACGAAGGAGAAAC AGCAGACAAGGAACTGGGAGCACTGCTGAAGGGAGTCG CAGGAAACGCACACGCACTGCAGACAGGAGACTTCAGA ACACCGGCAGAACTGGCACTGAACAAGTTCGAAAAGGA ATCGGGACACATCAGAAACCAGAGATCGGACTACTCGC ACACATTCTCGAGAAAGGACCTGCAGGCAGAACTGATC CTGCTGTTCGAAAAGCAGAAGGAATTCGGAAACCCGCA CGTCTCGGGAGGACTGAAGGAAGGAATCGAAACACTGC TGATGACACAGAGACCGGCACTGTCGGGAGACGCAGTC CAGAAGATGCTGGGACACTGCACATTCGAACCGGCAGA ACCGAAGGCAGCAAAGAACACATACACAGCAGAAAGAT TCATCTGGCTGACAAAGCTGAACAACCTGAGAATCCTGG AACAGGGATCGGAAAGACCGCTGACAGACACAGAAAGA GCAACACTGATGGACGA ACCGTACAGAAAGTCGAAGCT 67

- 209 047139

GACATACGCACAGGCAAGAAAGCTGCTGGGACTGGAAG ACACAGCATTCTTCAAGGGACTGAGATACGGAAAGGAC AACGCAGAAGCATCGACACTGATGGAAATGAAGGCATA CCACGCAATCTCGAGAGCACTGGAAAAGGAAGGACTGA AGCAAAGAAGTCGCCGCTGAACCTGTCGCCGGAACTG CAGGACGAAATCGGAACAGCATTCTCGCTGTTCAAGAC AGACGAAGACATCACAGGA AGACTGAAGGACAGAATCC AGCCGGAAATCCTGGAAGCACTGCTGAAGCACATCTCGT TCGACAAGTTCGTCCAGATCTCGCTGAAGGCACTGAGAA GAATCGTCCCGCTGATGGAACAGGGAAAGAGATACGAC GAAGCATGCGCAGAAATCTACGGAGACCACTACGGAAA GAAGAACACAGAAGAAAAGATCTACCTGCCGCCGATCC CGGCAGACGAAATCAGAAACCCGTCGTCCTGA GAGCA CTGTCGCAGGCAAGAAAGGTCATCAACGGAGTCGTCAG AAGATACGGATCGCCGGCAAGAATCCACATCGAAACAG CAAGAGAAGTCGGAAAGTCGTTCAAGGACAGAAAGGAA ATCGAAAAGAGACAGGAAGAAAACAGAAAGGACAGAG AAAAGGCAGCAGCAAAGTTCAGAGAATACTTCCCGAAC TTCGTCGGAGAACCGAAGTCGAAGGACATCCTGAAGCT GAGACTGTAC GAACAGCAGCACGGAAAGTGCCTGTACT CGGGAAAGGAAATCAACCTGGGAAGACTGAACGAAAAG GGATACGTCGAAATCGACCACGCACTGCCGTTCTCGAGA ACATGGGACGACTCGTTCAACAACAAGGTCCTGGTCCTG GGATCGGAAAACCAGAACAAGGGAAACCAGACACCGTA CGAATACTTCAACGGAAAGGACAACTCGAGAGAATGGC AGGAATTCAAGGCAAGAGTCGAAA CATCGAGATTCCCG AGATCGAAGAAGCAGAGAATCCTGCTGCAGAAGTTCGA CGAAGACGGATTCAAGGAAAGAAACCTGAACGACACAA GATACGTCAACAGATTCCTGTGCCAGTTCGTCGCAGACA GAATGAGACTGACAGGAAAGGGAAAGAAGAGAGTCTTC GCATCGAACGGACAGATCACAAACCTGCTGAGAGGATT CTGGGGACTGAGAAAGGTCAGAGCAGAAAACGACAGAC ACC ACGCACTGGACGCAGTCGTCGTCGCATGCTCGACAG TCGCAATGCAGCAGAAGATCACAAGATTCGTCAGATAC AAGGAAATGAACGCATTCGACGGAAAGACAATCGACAA

- 210 047139

GGAAACAGGAGAAGTCCTGCACCAGAAGACACACTTCC CGCAGCCGTGGGAATTCTTCGCACAGGAAGTCATGATCA GAGTCTTCGGAAAGCCGGACGGAAAGCCGGAATTCGAA GAAGCAGACACACTGGAAAAGCTGAGAACACTGCTGGC AGAAAAGCTGTCGTCGAGACCGGAAGCAGTCCACGAAT ACGTCACACCGCTGTTCGTCTCGAGAGCACCGAACAGAA TCGGGACAGGGACACATGGAAACAGTCAAGTCG GCAAAGAGACTGGACGAAGGAGTCTCGGTCCTGAGAGT CCCGCTGACACAGCTGAAGCTGAAGGACCTGGAAAAGA TGGTCAACAGAGAAAGAGAACCGAAGCTGTACGAAGCA CTGAAGGCAAGACTGGAAGCACACAAGGACGACCCGGC AAAGGCATTCGCAGAACCGTTCTACAAGTACGACAAGG CAGGAAACAGAACACAG CAGGTCAAGGCAGTCAGAGTC GAACAGGTCCAGAAGACAGGAGTCTGGGTCAGAAACCA CAACGGAATCGCAGACAACGCAACAATGGTCAGAGTAG ACGTCTTCGAAAAGGGAGACAAGTACTACCTGGTCCCG ATCTACTCGTGGCAGGTCGCAAAGGGAATCCTGCCGGAC AGAGCAGTCGTCCAGGAAAGGACGAAGAAGACTGGCA GCTGATCGACGACTCGTTCAACTTCAAG TTCTCGCTGCA CCCGAACGACCTGGTCGAAGTCATCACAAAGAAGGCAA GAATGTTCGGATACTTCGCATCGTGCCACAGAGGAACAG GAAACATCAACATCAGAATCCACGACCTGGACCACAAG ATCGGAAAGAACGGAATCCTGGAAGGAATCGGAGTCAA GACAGCACTGTCGTTCCAGAAGTACCAGATCGACGAACT GGGAAAGGAAATCAGACCGTGCAGACTGAAGAAGAGAC CGCCGGTCAGATCCGGAAAGAGAACAGCAGACGGATCG GAATTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAATG ATAGCTAGCTCGAGTCTAGAGGGCCCGTTTTAAACCCGCT GATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTG TTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGG TGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAAT CATTGTCTGAGTAGGTGTCATTCTATTCTGGG GGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGG GAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTC TATGG

- 211 047139

Amino acid sequence of Cas9 Neisseria meningitidis MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLG VRVFERAEVPKTGDSLAMARRLARSVRRLLTRRRAHRLLRT RRLLKREGVLQAANFDENGLIKSLPNTPWQLRAAALDRKL TPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGV AGNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSH t DRIQP EILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAE IYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVIN GVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDR EKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSG KEINLGRLNEKGY VEIDHALPFSRTWDDSFNNKVLVLGSE NQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKK QRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTG KGKKRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAV VVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGEVLH PWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRT LLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVK SAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEAL KARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQ VQKTGVWVRNHNGIADNATMVRVDVFEKGDKYY LVPIYS WQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDL VEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVRSGKRTAD GSEFESPKKKRKVE 68 Guide RNA G390 mG*mC*mC*GAGUCUGGAGAGCUGCAGUUUUAGAmGmC mU mAmGmAmAmAmU mAmGmC A AGUU A A A AU AAGGCU AGU CC GUU AU C AmAmCmU mU mGmAmAmAmAmAmGmU mGmGmCmAmCmCmGmAmGmUmCmGmGmUmG mCmU*m U*mU*mU 69 Guide RNA G502 mA*mC*mA*CAAAUACCAGUCCAGCGGUUUUAGAmGmC mU mAmGmAmAmAmU mAmGmC A AGUU A A A AU AAGGCU 70

- 212 047139

AGU СС GUU AU WITH AmAmCmU mU mGmAmAmAmAmAmGmU mGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*m U*mU*mU Guide RNA G509 mA*mA*mA*GUUCUAGAUGCCGUCCGGUUUUAGAmGmC mU mAmGmAmAmAmU mAmGmC A AGUU A A A AU AAGGCU AGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmU mGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmC mU*m U*mU*mU 71 Guide RNA G534 mA*mC*mG*CAAAUAUCAGUCCAGCGGUUUUAGAmGmC mU mAmGmAmAmAmU mAmGmC A AGUU A A A AU AAGGCU AGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmU mGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmC mU*m U*mU*mU 72

* = PS connection; 'm' = 2'-O-Me nucleotide.

Primer sequences for mouse NGS G000282.

Direct primer:

CACTCTTTCCCTACACGACGCTCTTCCGATCTGTTTTGTTCCAGAGTCTATCACCG

Reverse Primer:

GGAGTTCAGACGTGTGCTCTTCCGATCTACACGAATAAGAGCAAAATGGGAAC

Primer sequences for rat NGS G000390

Direct primer:

CACTCTTTCCCTACACGACGCTCTTCCGATCTTGCATTTCATGAGACCGAAAACA

Reverse Primer:

GGAGTTCAGACGTGTGCTCTTCCGATCTGCTACAGTAGAGCTGTACATAAAACTT GFP sequence:

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGA GACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCG CGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAG ATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAG AAAATACCGCATCAGGCGCCAT TCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGC GATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCA AGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGAC GGCCAGTGAATTCTAATACGACTCACTATAGGGTCCCGCAGTCGGCGTCCAGCGGCT CTGCTTGTTCGTGTGTGTCGTTGCAGGCCTTATT CGGATCCATGGTGAGCAAGGG CGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAG CTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTC GTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGAC CACATGAAG

- 213 047139

CAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATC TTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGA CACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACA TCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCC GACAAGCAGAAGAACGG CATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGC CCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGA CCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGA TCACTCTCGGCATGGACGAGCTGTACAAGTAATAGGAATTAT A AAAAAAAAAAAAAAAAAA AAAAAATCTAGACTTAAGCTTGATGAGCTCTAGCTTGGCGTAATCATGGTCATAGCT GTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAG CATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTT GCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAAT CG GCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCT CACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA GGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAG CAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGTTTGCTGGCGTTTTTC CATAGGCTCCGCCCCCCTGACG AGCATCACAAAAATCGACGCTCAAGTCAGAGGTG GCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTC GGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCC CCCGTTCAGCCCGACCGCTGCGC CTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCT GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC TCTTGATCCGGCA AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTC ACCTAGATCCTTTTAAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG TAAACT TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATC TGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATAC GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCA CCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAG TGGTCCTGCAACTTTATCCG CCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAG

- 214 047139

AGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCAT CGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCA AGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTCGGTCCT CCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGT ACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGG CGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTG GAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT CGATGTAACCCACTCGTG CACCCAACTGATCTTCAGCATCTTTTTACTTTCACCAGCGT TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATTATTGAAGCATTTATC AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC TAAGAAACCATTA TTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCG

Claims (48)

1. Pharmaceutical composition of lipid nanoparticles containing: An RNA component, wherein the RNA component comprises (i) an mRNA encoding an RNA-directed DNA binding agent and (ii) a gRNA nucleic acid; and a lipid component, wherein the lipid component comprises: 50-60 mol.% aminolipid; 8-10 mol.% neutral lipid; And 2.5-4 mol.% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, the aminolipid is represented by the following structural formula: wherein R ¹ and R ² are each independently C ₄ -C ₁₂ alkyl and the N/P ratio of the LNP composition is 6. 2. LNP pharmaceutical composition containing: An RNA component, wherein the RNA component comprises (i) an mRNA encoding an RNA-directed DNA binding agent and (ii) a gRNA nucleic acid; and a lipid component, wherein the lipid component comprises: 50-60 mol.% aminolipid; 27-39.5 mol% auxiliary lipid; 8-10 mol.% neutral lipid; And 2.5-4 mol.% PEG lipid, aminolipid represented by the following structural formula: where R ¹ and R ² each independently represent C.|-C| ₂ ii.n<Hi, and wherein the N/P ratio of the LNP composition is 5-7. 3. The composition according to claim 6, wherein the lipid component contains: 50-60 mol.% aminolipid; 5-10 mol.% neutral lipid; And 2.5-4 mol.% PEG lipid. 4. LNP pharmaceutical composition containing: An RNA component, wherein the RNA component comprises (i) an mRNA encoding an RNA-directed DNA binding agent and (ii) a gRNA nucleic acid; and a lipid component, wherein the lipid component comprises: 40-60 mol.% aminolipid; - 215 047139 5-15 mol.% neutral lipid; And 2.5-4 mol.% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, the aminolipid is represented by the following structural formula: where R ¹ and R ² are each independently C ₄ -C ₁₂ alkyl, and the N/P ratio of the LNP composition is 6. 5. LNP pharmaceutical composition containing: An RNA component, wherein the RNA component comprises (i) an mRNA encoding an RNA-directed DNA binding agent and (ii) a gRNA nucleic acid; and a lipid component, wherein the lipid component comprises: 50-60 mol.% aminolipid; 5-15 mol.% neutral lipid; And 1.5-10 mol.% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, the aminolipid is represented by the following structural formula: where R ¹ and R ² are each independently C ₄ -C ₁₂ alkyl, and the N/P ratio of the LNP composition is 6. 6. LNP pharmaceutical composition containing: An RNA component, wherein the RNA component comprises (i) an mRNA encoding an RNA-directed DNA binding agent and (ii) a gRNA nucleic acid; and a lipid component, wherein the lipid component comprises: 40-60 mol.% aminolipid; less than 10 mol.% neutral lipid; And 1.5-10 mol.% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, the aminolipid is represented by the following structural formula: where R ¹ and R ² are each independently C ₄ -C ₁₂ alkyl, and the N/P ratio of the LNP composition is 5-7. 7. The composition according to claim 6, wherein the lipid component contains: less than 1 mol.% neutral lipid. 8. LNP pharmaceutical composition containing: An RNA component, wherein the RNA component comprises (i) an mRNA encoding an RNA-directed DNA binding agent and (ii) a gRNA nucleic acid; and a lipid component, wherein the lipid component comprises: 50-60 mol.% aminolipid; 8-10 mol.% neutral lipid; And 2.5-4 mol.% PEG lipid, wherein the remainder of the lipid component is an auxiliary lipid, the aminolipid is represented by the following structural formula: - 216 047139 where R ¹ and R ² are each independently C ₄ -C ₁₂ alkyl, and the N/P ratio of the LNP composition is 5-7. 9. The composition according to any of the preceding claims, wherein the RNA-guided DNA binding agent is a Cas nuclease. 10. The composition of any one of the preceding claims, wherein the RNA component comprises a class 2 Cas nuclease mRNA, such as a Cas9 nuclease mRNA. 11. The composition according to claim 9 or 10, wherein the mRNA is modified mRNA. 12. The composition according to any of the preceding claims, wherein the mRNA contains an open reading frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has a uridine content ranging from a minimum uridine content to 150% of the minimum uridine content or an open frame frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has a uridine dinucleotide content ranging from a minimum uridine dinucleotide content to 150% of a minimum uridine dinucleotide content. 13. The composition according to any of the preceding claims, wherein the mRNA contains a sequence with at least 90% identity to any of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65 or 66 and wherein the mRNA contains an open reading frame encoding an RNA-guided DNA binding agent. 14. The composition according to any of the preceding claims, wherein the gRNA nucleic acid is gRNA. 15. The composition according to any of the preceding claims, wherein the RNA component comprises Cas class 2 nuclease mRNA and gRNA. 16. The composition of claim 14 or 15, wherein the gRNA nucleic acid is or encodes a double guide RNA (dgRNA) or sgRNA. 17. The composition according to any one of claims 14-16, in which the gRNA contains a modification, such as a modification selected from 2'-O-methyl (2'-O-Me)-modified nucleotide, phosphorothioate (PS) bond between nucleotides; and 2'-fluoro(2'^)-modified nucleotide. 18. The composition of claim 17, wherein the gRNA contains a modification on one or more of the first five nucleotides at the 5' end, or the gRNA contains a modification on one or more of the last five nucleotides at the 3' end. 19. The composition according to claim 17 or 18, wherein the gRNA contains PS bonds between the first four nucleotides or the gRNA contains PS bonds between the last four nucleotides. 20. The composition according to any one of claims 17 to 19, additionally containing 2'-O-Me-modified nucleotides in the first three nucleotides at the 5'-end or 2'-O-Me-modified nucleotides in the last three nucleotides at the 3'-end end. 21. The composition according to any one of claims 14-20, in which gRNA and mRNA Cas-nucleases of class 2 are present in a mass ratio of from 10:1 to 1:10, from 5:1 to 1:5, from 3:1 to 1 :1 or from 2:1 to 1:1. 22. The composition according to any one of claims 14-20, in which gRNA and mRNA Cas-nuclease class 2 are present in a mass ratio of 2:1 or in a mass ratio of 1:1. 23. The composition according to any of the preceding claims, further containing at least one template nucleic acid. 24. The composition according to any of the preceding claims, wherein the lipid component contains 3 mol.% PEG lipid. 25. The composition according to any of the preceding claims, wherein the lipid component contains 50 or 55 mol.% aminolipid. 26. The composition according to any of the preceding claims, in which the lipid component contains 47-53, 48-53 or 53-57 mol.% aminolipid. 27. The composition according to any of the preceding paragraphs, in which the N/P ratio is 6±1. 28. The composition according to any of the preceding paragraphs, in which the N/P ratio is 6 ± 0.5. 29. The composition according to any of the preceding claims, wherein each of R ¹ and R ² independently represents ^-^alkyl. 30. The composition according to any one of claims 1 to 28, wherein R ¹ and R ² are each independently C ₅ C 1 ₀ alkyl. 31. Composition according to any one of claims 1-28, where the acetal analogue is selected from C4, C5, C6, C7, C9, C10, - 217 047139 C11 and C12 analogues. 32. The composition according to any one of claims 1 to 28, wherein the aminolipid is Lipid A, wherein Lipid A is represented by the following structural formula: 33. The composition according to any of the preceding claims, wherein the auxiliary lipid is selected from cholesterol, 5-heptadecylresorcinol and cholesterol hemisuccinate. 34. The composition according to any of the preceding claims, wherein the auxiliary lipid is cholesterol. 35. The composition according to any of the preceding claims, wherein the neutral lipid is selected from dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3phosphocholine (PLPC) , 1,2-diarachidoyl-sn-glycero-3-phosphatidylcholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3 -phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE ), lysophosphatidylethanolamine and combinations thereof. 36. The composition according to any of the preceding claims, wherein the neutral lipid is DSPC or DPPC. 37. The composition according to any of the preceding claims, wherein the neutral lipid is DSPC. 38. The composition according to any one of claims 1 to 36, wherein the neutral lipid is DPPC. 39. The composition according to any one of claims 1 to 35, wherein the neutral lipid is dimyristoylphosphatidylethanolamine (DMPE). 40. The composition according to any of the preceding claims, wherein the PEG lipid contains dimyristoylglycerol (DMG) and/or the PEG lipid contains PEG-2k. 41. The composition according to any of the preceding claims, wherein the PEG lipid is PEG-DMG. 42. The composition according to claim 41, wherein the PEG-DMG is PEG2k-DMG. 43. The composition according to claim 40, wherein the PEG lipid is PEG2k-DMG. 44. The composition according to any one of claims 1 to 39, where the PEG lipid is selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglycerol, PEG -dimyristyl glycamide, PEG-dipalmitoyl glycamide, PEG-distearoyl glycamide, PEG-cholesterol (1-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'dioxaoctanyl]carbamoyl-[omega]- methyl poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxybenzyl[omega]-methyl-poly(ethylene glycol)ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE), 1,2-distearoyl-sn -glycerol methoxypolyethylene glycol (PEG2k-DSG), polyethylene glycol-2000-dimethacrylate (PEG2k-DMA) and 1,2 distearyloxypropyl-3-amine-N[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). 45. A method of editing genes or producing a genetically engineered cell, comprising contacting the cell with the LNP composition of any one of the preceding claims. 46. The method of claim 45, wherein the LNP composition is administered at least twice, such as 2-5 times. 47. The method of claim 46, wherein the editing improves with repeated administration. 48. The method according to any one of claims 45-47, further comprising introducing at least one template nucleic acid into the cell.