CN110835630A - A high-efficiency sgRNA and its application in gene editing - Google Patents

Abstract

The invention provides an efficient sgRNA and application thereof in gene editing. The sgRNA is shown as formula I: an RNA-engineered sgRNA backbone transcribed from the target sequence (formula I); the modified sgRNA framework is an RNA molecule obtained by inserting an RNA fragment A between 14 th and 15 th positions of the sgRNA framework and inserting an RNA fragment B between 18 th and 19 th positions of the sgRNA framework; the RNA segment A and the RNA segment B are reversely complementary; the sizes of the RNA fragment A and the RNA fragment B are both 3 nt; the sgRNA framework is an RNA molecule shown in a sequence 9. Experiments prove that: the modified sgRNA can obviously improve the C.T base replacement efficiency of a Cytosine Base Editor (CBE), and the highest efficiency can reach 86.4%.

Description

A high-efficiency sgRNA and its application in gene editing

technical field

The invention belongs to the field of biotechnology, in particular to an efficient sgRNA and its application in gene editing.

Background technique

CRISPR-Cas9 technology has become a powerful genome editing method, which is widely used in many tissues and cells. The CRISPR/Cas9 protein-RNA complex is positioned on the target through the guide RNA (guide RNA), and the cleavage produces a DNA double-strand break (dsDNA break, DSB), and then the organism will instinctively initiate the DNA repair mechanism to repair the DSB. There are generally two repair mechanisms, one is non-homologous end joining (NHEJ) and the other is homologous recombination (homology-directed repair, HDR). Usually NHEJ is in the majority, so the random indels (insertions or deletions) generated by repair are much higher than with precise repair. For base-accurate substitutions, the application of base-accurate substitutions using HDR is greatly limited due to the low efficiency of HDR and the need for DNA templates.

In 2016, the laboratories of David Liu and Akihiko Kondo independently reported two different types of cytosine base editors (CBE), using two different cytidine deaminase rAPOBEC1 (rat APOBEC1) and PmCDA1 (activation-induced cytidine deaminase (AID) ortholog from sea lamprey), the principle is to directly edit a single cytosine (Cytosine, C) base by using cytidine deaminase, instead of generating DSB and initiation of HDR repair greatly improved the base editing efficiency of C replacement with Thymine (T). Specifically, dead Cas9 (dCas9) or the Cas9 nickase (Cas9n) together with rAPOBEC1 or PmCDA1 is localized to the target through sgRNA, and rAPOBEC1 or PmCDA1 catalyzes the deamination of C on unpaired single-stranded DNA into uracil ( Uracil, U), through DNA repair, U is paired with adenine (A), and through DNA replication, T is finally paired with A, thereby realizing the conversion of C to T. Among the editors tested, the SpCas9n(D10A)&rAPOBEC1/PmCDA1&UGI base editing system (which contains a uracil DNA glycosylase inhibitor (UGI)) had a higher average mutation rate for two reasons: one UGI can inhibit uracil DNA glycosylase (UDG) to catalyze the removal of U in DNA, and the second is SpCas9n (D10A) nicking on the non-editing strand, inducing eukaryotic mismatch repair mechanism or long-patch BER (base -excision repair) repair mechanism, which promotes more preferential repair of U:G mismatches into U:A. In order to improve work efficiency and reduce work costs, the improvement of C·T base replacement efficiency has always been the research direction of base editing systems for animal and plant genomes.

Cas9 (SaCas9) derived from Staphylococcus aureus is a SpCas9 homolog that recognizes NNGRRT PAM, and SaCas9 variant SaKKH recognizes the more extensive NNNRRT PAM, both of which were developed into effective CBEs, greatly expanding CBE The range of editable C in animal and plant genomes. There is no research report on improving the C·T base substitution efficiency of SaKKH-related CBE by modifying the structure of the corresponding sgRNA of SaCas9 (SaCas9 sgRNA).

SUMMARY OF THE INVENTION

The purpose of the present invention is to improve the C·T base substitution efficiency of a cytosine base editor (CBE).

In order to achieve the above purpose, the present invention provides a kit of reagents, the kit includes sgRNA or biological material related to the sgRNA, Cas9 nuclease or biological material related to the Cas9 nuclease, cytosine deaminase or biological material associated with said cytosine deaminase;

the sgRNA targeting target sequence;

Described sgRNA is as shown in formula I: the RNA-engineered sgRNA backbone (formula I) transcribed by the target sequence;

The modified sgRNA backbone is an RNA molecule obtained by inserting RNA fragment A between positions 14-15 of the sgRNA backbone, and inserting RNA fragment B between positions 18-19 of the sgRNA backbone;

The RNA fragment A is reverse complementary to the RNA fragment B;

The size of the RNA fragment A and the RNA fragment B are both 3nt;

The sgRNA backbone is m1) or m2) or m3):

m1) the RNA molecule shown in sequence 9;

m2) the RNA molecule shown in m1) has undergone the substitution and/or deletion and/or addition of one or several nucleotides and has the same function;

m3) An RNA molecule that is 75% or more identical to the nucleotide sequence defined by m1) or m2) and has the same function.

In the above-mentioned complete set of reagents, the modified sgRNA backbone is n1) or n2) or n3):

n1) RNA molecule shown in sequence 10;

n2) the RNA molecule shown in n1) has undergone the substitution and/or deletion and/or addition of one or several nucleotides and has the same function;

n3) An RNA molecule having 75% or more identity to the nucleotide sequence defined by n1) or n2) and having the same function.

In the above reagent kit, the Cas9 nuclease may be a protein such as SaKKHn or SaCas9 or SaKKH-HF or SaCas9-HF. In a specific embodiment of the present invention, the Cas9 nuclease is specifically SaKKHn protein.

Said SaKKHn protein is E1) or E2) or E3):

E1) the amino acid sequence is the protein shown in sequence 2;

E2) The amino acid sequence shown in SEQ ID NO: 2 in the sequence listing is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and has the same function as a protein;

E3) a fusion protein obtained by linking a tag at the N-terminus or/and C-terminus of E1) or E2);

The biological material associated with the SaKKHn protein is any one of F1) to F5):

F1) a nucleic acid molecule encoding the SaKKHn protein;

F2) an expression cassette containing the nucleic acid molecule of F1);

F3) a recombinant vector containing the nucleic acid molecule described in F1) or a recombinant vector containing the expression cassette described in F2);

F4) a recombinant microorganism containing the nucleic acid molecule described in F1), or a recombinant microorganism containing the expression cassette described in F2), or a recombinant microorganism containing the recombinant vector described in F3);

F5) A transgenic cell line containing the nucleic acid molecule of F1), or a transgenic cell line containing the expression cassette of F2).

In the above reagent kit, the cytosine deaminase can be proteins such as human APOBEC3A, human AID, PmCDA1 or rAPOBEC1. In a specific embodiment of the present invention, the cytosine deaminase is specifically PmCDA1 protein.

The PmCDA1 protein is G1) or G2) or G3):

G1) the amino acid sequence is the protein shown in sequence 3;

G2) The amino acid sequence shown in SEQ ID NO: 3 in the sequence listing is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and has the same function as a protein;

G3) a fusion protein obtained by linking a tag to the N-terminus or/and C-terminus of G1) or G2);

The biological material related to the PmCDA1 protein is any one of H1) to H5):

H1) a nucleic acid molecule encoding the PmCDA1 protein;

H2) an expression cassette containing the nucleic acid molecule described in H1);

H3) a recombinant vector containing the nucleic acid molecule described in H1) or a recombinant vector containing the expression cassette described in H2);

H4) a recombinant microorganism containing the nucleic acid molecule described in H1), or a recombinant microorganism containing the expression cassette described in H2), or a recombinant microorganism containing the recombinant vector described in H3);

H5) a transgenic cell line containing the nucleic acid molecule of H1), or a transgenic cell line containing the expression cassette of H2).

In the above-mentioned complete set of reagents, the sgRNA can be tRNA-sgRNA;

Described tRNA-sgRNA is as shown in formula I: tRNA-the RNA-remodeled sgRNA backbone (formula I) transcribed by the target sequence;

The tRNA is 1) or 2) or 3):

1) the RNA molecule obtained by replacing T in the 474th-550th position of sequence 1 with U;

2) The RNA molecule shown in 1) has been replaced and/or deleted and/or added by one or several nucleotides and has the same function as the RNA molecule;

3) An RNA molecule having 75% or more identity with the nucleotide sequence defined in 1) or 2) and having the same function.

The above-mentioned complete set of reagents can also include UGI protein or biological material related to the UGI protein;

The UGI protein is I1) or I2) or I3):

I1) the amino acid sequence is the protein shown in sequence 4;

12) The amino acid sequence shown in SEQ ID NO: 4 in the sequence listing is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and has the same function as a protein;

I3) a fusion protein obtained by linking a tag at the N-terminus or/and C-terminus of I1) or I2);

The biological material associated with the UGI protein is any one of J1) to J5):

J1) a nucleic acid molecule encoding the UGI protein;

J2) an expression cassette containing the nucleic acid molecule of J1);

J3) a recombinant vector containing the nucleic acid molecule described in J1), or a recombinant vector containing the expression cassette described in J2);

J4) a recombinant microorganism containing the nucleic acid molecule described in J1), or a recombinant microorganism containing the expression cassette described in J2), or a recombinant microorganism containing the recombinant vector described in J3);

J5) A transgenic cell line containing the nucleic acid molecule of J1), or a transgenic cell line containing the expression cassette of J2).

In order to facilitate the purification of the proteins in E1), G1) and I1), the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 can be linked as shown in the table below Tag of.

Sequence of tables, tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The above-mentioned proteins in E2), G2) and I2) are proteins that are 75% or more identical to the amino acid sequence of the protein represented by SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and have the same function. Having 75% or more identity is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical .

The proteins in the above E2), G2), and I2) can be artificially synthesized, or their encoding genes can be synthesized first, and then biologically expressed.

The genes encoding the proteins in the above E2), G2), and I2) can be obtained by combining the 3013-6225th position of sequence 1 (the protein shown in coding sequence 2) and the 6511-7134 position of sequence 1 (coding sequence 3). protein), 7156-7452 of SEQ ID NO: 1 (encoding the protein shown in SEQ ID NO: 4) in the DNA sequence with deletion of one or several amino acid residues in codons, and/or one or several base pairs missense mutation, and/or linking the coding sequences of the tags shown in the table above at its 5' and/or 3' ends.

Further, the nucleic acid molecule of F1) is f1) or f2) or f3):

f1) The cDNA molecule or DNA molecule shown in position 3013-6225 of sequence 1 in the sequence listing;

f2) has 75% or more identity with the nucleotide sequence defined in f1), and encodes a cDNA molecule or DNA molecule of said SaKKHn;

f3) hybridizes under stringent conditions to the nucleotide sequence defined in f1) or f2) and encodes a cDNA molecule or DNA molecule of said SaKKHn;

H1) the nucleic acid molecule is h1) or h2) or h3):

h1) The cDNA molecule or DNA molecule shown in position 6511-7134 of sequence 1 in the sequence listing;

h2) has 75% or more identity with the nucleotide sequence defined in h1), and encodes a cDNA molecule or DNA molecule of said PmCDA1;

h3) hybridizes to a nucleotide sequence defined by h1) or h2) under stringent conditions, and encodes a cDNA molecule or DNA molecule of said PmCDA1;

J1) The nucleic acid molecule is j1) or j2) or j3):

j1) The cDNA molecule or DNA molecule shown in position 7156-7452 of sequence 1 in the sequence listing;

j2) a cDNA molecule or DNA molecule that is 75% or more identical to the nucleotide sequence defined in j1) and encodes the UGI;

j3) a cDNA molecule or a DNA molecule that hybridizes under stringent conditions to a nucleotide sequence defined in j1) or j2) and encodes said UGI.

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the SaKKHn or the PmCDA1 or the UGI of the present invention using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity to the nucleotide sequence of the SaKKHn or the PmCDA1 or the UGI of the present invention, as long as they encode the SaKKHn or the PmCDA1 or the UGI. The UGI described above and having the same function are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or more, or 85% or more, or 90% or more of the nucleotide sequence of the protein consisting of the amino acid sequences shown in the coding sequences 2, 3, and 4 of the present invention, or nucleotide sequences of 95% or greater identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The stringent conditions were hybridization in a solution of 2×SSC, 0.1% SDS at 68°C and washing the membrane twice for 5 min each, and hybridization in a solution of 0.5×SSC, 0.1% SDS at 68°C. And wash the membrane twice for 15 min each time; or, in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS, hybridize and wash the membrane at 65°C.

The above-mentioned 75% or more identity may be 80%, 85%, 90% or more than 95% identity.

F2) The described expression cassette (SaKKHn gene expression cassette) containing the nucleic acid molecule encoding SaKKHn protein refers to the DNA capable of expressing SaKKHn protein in the host cell. Includes a terminator that terminates transcription of the SaKKHn gene. Further, the expression cassette may also include enhancer sequences. The recombinant vector containing the SaKKHn gene expression cassette can be constructed by using the existing expression vector.

H2) The described expression cassette (PmCDA1 gene expression cassette) containing the nucleic acid molecule encoding the PmCDA1 protein refers to the DNA capable of expressing the PmCDA1 protein in the host cell. Includes a terminator that terminates transcription of the PmCDA1 gene. Further, the expression cassette may also include enhancer sequences. The recombinant vector containing the PmCDA1 gene expression cassette can be constructed by using the existing expression vector.

The expression cassette (UGI gene expression cassette) containing the nucleic acid molecule encoding UGI protein described in J2) refers to the DNA capable of expressing the UGI protein in the host cell. Includes a terminator that terminates transcription of the UGI gene. Further, the expression cassette may also include enhancer sequences. The recombinant vector containing the UGI gene expression cassette can be constructed by using the existing expression vector.

The vector may be a plasmid, cosmid, phage or viral vector. In a specific embodiment of the present invention, the recombinant vector is specifically SaKKHn-pBE+3bp-1 recombinant expression vector, SaKKHn-pBE+3bp-2 recombinant expression vector, SaKKHn-pBE+3bp-3 recombinant expression vector, SaKKHn- pBE+3bp-4 recombinant expression vector, SaKKHn-pBE+3bp-5 recombinant expression vector, SaKKHn-pBE+3bp-6 recombinant expression vector or SaKKHn-pBE+3bp-7 recombinant expression vector.

The nucleotide sequence of the SaKKHn-pBE+3bp-1 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the SaKKHn-pBE-1 recombinant expression vector sequence with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6 , and keep other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+3bp-2 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the SaKKHn-pBE-2 recombinant expression vector sequence with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6 , and keep other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+3bp-3 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the SaKKHn-pBE-3 recombinant expression vector sequence with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6, The sequence obtained after keeping other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+3bp-4 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the SaKKHn-pBE-4 recombinant expression vector sequence with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6, The sequence obtained after keeping other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+3bp-5 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the SaKKHn-pBE-5 recombinant expression vector sequence with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6, The sequence obtained after keeping other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+3bp-6 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the SaKKHn-pBE-6 recombinant expression vector sequence with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6, The sequence obtained after keeping other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+3bp-7 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the SaKKHn-pBE-7 recombinant expression vector sequence with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6, The sequence obtained after keeping other sequences unchanged.

The microorganism can be yeast, bacteria, algae or fungi. Wherein, the bacteria can be Agrobacterium, such as Agrobacterium EHA105. In a specific embodiment of the present invention, the recombinant microorganism specifically contains the SaKKHn-pBE+3bp-1 recombinant expression vector, the SaKKHn-pBE+3bp-2 recombinant expression vector, the SaKKHn-pBE+3bp- 3 recombinant expression vector, described SaKKHn-pBE+3bp-4 recombinant expression vector, described SaKKHn-pBE+3bp-5 recombinant expression vector, described SaKKHn-pBE+3bp-6 recombinant expression vector or described SaKKHn-pBE+ 3bp-7 recombinant expression vector of Agrobacterium EHA105.

The transgenic cell line does not include propagation material.

The above-mentioned complete set of reagents has the following purposes:

X1) Editing of genome target sequences of organisms or biological cells;

X2) Preparation of edited products of organism or biological cell genome target sequence;

X3) Improve the editing efficiency of the genome target sequence of the organism or biological cell;

X4) Prepare a product that improves the editing efficiency of the genome target sequence of an organism or a biological cell.

The sgRNA or the modified sgRNA backbone in the above-mentioned complete set of reagents also belong to the protection scope of the present invention.

In order to achieve the above-mentioned purpose, the present invention also provides a new use of the above-mentioned complete set of reagents or sgRNA or modified sgRNA backbone.

The invention provides the application of the above-mentioned complete set of reagents or sgRNA or the new application of the modified sgRNA backbone in any one of X1)-X4):

X1) Editing of genome target sequences of organisms or biological cells;

X2) Preparation of edited products of organism or biological cell genome target sequence;

X3) Improve the editing efficiency of the genome target sequence of the organism or biological cell;

X4) Prepare a product that improves the editing efficiency of the genome target sequence of an organism or a biological cell.

In order to achieve the above-mentioned purpose, the present invention finally provides the method described in Y1) or Y2):

Y1) a method for editing a genome target sequence or a method for improving the editing efficiency of an organism or biological cell genome target sequence, comprising expressing the above-mentioned sgRNA, the above-mentioned Cas9 nuclease, and the above-mentioned cytosine deaminase in the organism or biological cell, Realize the editing of the genome target sequence; the sgRNA targets the target sequence;

Y2) A method for preparing biological mutants, comprising the steps of: editing the genome of an organism according to the method described in Y1) to obtain biological mutants.

In the above method, in the Y1), the sgRNA is the above-mentioned tRNA-sgRNA, and the tRNA-sgRNA obtained after the DNA molecule transcribing the tRNA-sgRNA is transcribed is an immature RNA precursor, and the tRNA in the RNA precursor is Mature RNA is obtained after being cleaved by two enzymes (RNase P and RNase Z). There are as many targets in a recombinant expression vector as there are independent mature RNAs, and each mature RNA is composed of the RNA transcribed by the target sequence and the sgRNA backbone in turn, or the target sequence is composed of the target sequence in turn. The individual base composition of the dot sequence transcribed RNA, the sgRNA backbone, and the tRNA residues.

In the above method, the Y1) further includes the step of expressing UGI in the organism or biological cell, and the number of the UGI can be one or two or more. In a specific embodiment of the present invention, the number of the UGI is specifically one.

Further, the method for expressing the above-mentioned sgRNA, the above-mentioned Cas9 nuclease, the above-mentioned cytosine deaminase and the above-mentioned UGI in the organism or biological cell is to transcribe the encoding gene of the Cas9 nuclease, the DNA molecule of the sgRNA, the The cytosine deaminase-encoding gene and the UGI-encoding gene are introduced into an organism or biological cell.

Further, the coding gene of the Cas9 nuclease, the DNA molecule transcribing the sgRNA, the coding gene of the cytosine deaminase and the coding gene of the UGI are introduced into the organism or biological cell through a recombinant expression vector.

The encoding gene of the Cas9 nuclease, the DNA molecule transcribing the sgRNA, the encoding gene of the cytosine deaminase and the encoding gene of the UGI can be introduced into the organism or biological cell through the same recombinant expression vector, or Two or more recombinant expression vectors can be co-introduced into organisms or biological cells.

In a specific embodiment of the present invention, the coding gene of the Cas9 nuclease, the DNA molecule transcribing the sgRNA, the coding gene of the cytosine deaminase and the coding gene of the UGI are introduced through the same recombinant expression vector within an organism or biological cell. The recombinant expression vector contains an expression cassette A and an expression cassette B; the expression cassette A expresses the above-mentioned sgRNA, and the expression cassette B expresses a fusion protein composed of the above-mentioned Cas9 nuclease, the above-mentioned cytosine deaminase and the above-mentioned UGI.

The recombinant expression vector is specifically the SaKKHn-pBE+3bp-1 recombinant expression vector, the SaKKHn-pBE+3bp-2 recombinant expression vector, the SaKKHn-pBE+3bp-3 recombinant expression vector, the SaKKHn- pBE+3bp-4 recombinant expression vector, the SaKKHn-pBE+3bp-5 recombinant expression vector, the SaKKHn-pBE+3bp-6 recombinant expression vector or the SaKKHn-pBE+3bp-7 recombinant expression vector.

In the above kit of reagents or applications or methods, the number of the target sequence may be one or two or more. The PAM sequence of the target sequence is NNNRRT.

In the above kit or application or method, the editing of the genomic target sequence is to mutate C in the target sequence to T. The C is C at any position in the target sequence.

In the above-mentioned complete set of reagents or applications or methods, the organism is S1) or S2) or S3) or S4):

S1) plants or animals;

S2) monocotyledonous or dicotyledonous plants;

S3) Poaceae;

S4) rice;

The biological cell is T1) or T2) or T3) or T4):

T1) plant cells or animal cells;

T2) monocotyledonous plant cells or dicotyledonous plant cells;

T3) Gramineae cells;

T4) Rice cells.

The present invention provides a modified sgRNA whose structure is shown in formula I: RNA-modified sgRNA backbone (formula I) transcribed by the target sequence; the modified sgRNA backbone is between positions 14-15 of the sgRNA backbone An RNA molecule obtained by inserting RNA fragment A between positions 18 and 19 of the sgRNA backbone, and inserting RNA fragment B between positions 18-19 of the sgRNA backbone; the RNA fragment A and the RNA fragment B are inversely complementary; the RNA fragment A and The size of the RNA fragment B is 3nt; the sgRNA backbone is the RNA molecule shown in sequence 9. It is proved by experiments that the modified sgRNA of the present invention can significantly improve the C·T base substitution efficiency of the cytosine base editor (CBE), up to 86.4%.

Description of drawings

Figure 1 shows the unmodified SaCas9 sgRNA structure and the modified SaCas9 sgRNA structure.

Figure 2 is a schematic structural diagram of a recombinant expression vector.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5'-terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3'-terminal nucleus of the corresponding DNA/RNA. Glycosides.

Primer pair T1 consists of primer T1-F: 5'-ttcaaattctaatccccaatcc-3' and primer T1-R: 5'-tcgtacctgtctgcaaccttg-3', which is used to amplify target T1.

Primer pair T2 consists of primer T2-F: 5'-gctttagatgatttgttacatttcgc-3' and primer T2-R: 5'-tgagttggtatggcaagaacaag-3', which is used to amplify target T2.

Primer pair T3 consists of primer T3-F: 5'-aacacggtcaccaacttcatc-3' and primer T3-R: 5'-acaacctggcttgctatatgc-3', which is used to amplify target T3.

Primer pair T4 consists of primer T4-F: 5'-tggatcggatatggacttctc-3' and primer T4-R: 5'-gaaatgaacaatcacctgagatctttg-3', which is used to amplify the targets T4 and T7.

Primer pair T5 consists of primer T5-F: 5'-cgagctacctgaagaacaactacc-3' and primer T5-R: 5'-cctcgattgcctgaaatttg-3', which is used to amplify target T5.

Primer pair T6 consists of primer T6-F: 5'-tgcgagctcgacaacatcatg-3' and primer T6-R: 5'-gacggcccatgtggaaacc-3', which is used to amplify target T6.

Primer pair T8 consists of primer T8-F: 5'-gacgcccatagtcgaggtc-3' and primer T8-R: 5'-ctctgctggatcaatgtcaatg-3', which is used to amplify target T8.

Primer pair T9 consists of primer T9-F: 5'-cctcatccaatcgactgacac-3' and primer T9-R: 5'-gtaattgtgcttggtgatggag-3', which is used to amplify target T9.

In the following examples, C·T base substitution means that C at any position in the target sequence is mutated to T.

C·T base substitution efficiency=number of positive T0 seedlings with C·T base substitutions/total number of positive T0 seedlings analyzed×100%.

Nipponbare Rice: References: Liang Weihong, Wang Gaohua, Du Jingyao, et al. Effects of sodium nitroprusside and its photolysis products on the growth of Nipponbare rice seedlings and the expression of five hormone marker genes [J]. Journal of Henan Normal University (Nature Edition), 2017 (2): 48-52.; Publicly available from Beijing Academy of Agriculture and Forestry.

Recovery medium: N6 solid medium containing 200 mg/L Timentin.

Screening medium: N6 solid medium containing 50 mg/L hygromycin.

Differentiation medium: N6 solid medium containing 2 mg/L KT, 0.2 mg/L NAA, 0.5 g/L glutamic acid, 0.5 g/L proline.

Rooting medium: N6 solid medium containing 0.2 mg/L NAA, 0.5 g/L glutamic acid, and 0.5 g/L proline.

Embodiment 1, the transformation of sgRNA backbone structure in SaCas9 sgRNA

The SaCas9 sgRNA structure is as follows: RNA-sgRNA backbone transcribed from the target sequence.

The sgRNA backbone structure in the SaCas9 sgRNA structure was modified, and the two sgRNA backbone structure modification methods are shown in Figure 1.

Original represents the unmodified SaCas9 sgRNA structure, the unmodified SaCas9 sgRNA is recorded as the Original sgRNA, and the sgRNA backbone in the Original sgRNA is recorded as the Original sgRNA backbone. The RNA sequence of the OriginalsgRNA backbone is as follows: GUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCGUCGAACUUGUUGGCGAGAU (sequence 9); Original sgRNA The DNA sequence of the backbone is as follows: GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGAT.

O+3bp represents the SaCas9 sgRNA structure obtained by adding 3 pairs of bases to the unmodified SaCas9 sgRNA structure, the modified SaCas9 sgRNA is denoted as +3bp sgRNA, and the sgRNA backbone in the +3bp sgRNA is denoted as +3bp sgRNA backbone, the RNA sequence of the +3bp sgRNA backbone is as follows: GUUUUAGUACUCUG CUG GAAA CAG CAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAU (SEQ ID NO: 10, the underlined sequence is an added 3 base pairs); the DNA sequence of the +3bp sgRNA backbone is shown in SEQ ID NO: 6.

O+8bp represents the SaCas9 sgRNA structure obtained by adding 8 pairs of bases to the unmodified SaCas9 sgRNA structure, the modified SaCas9 sgRNA is denoted as +8bp sgRNA, and the sgRNA backbone in the +8bp sgRNA is denoted as +8bp sgRNA backbone, the RNA sequence of the +8bp sgRNA backbone is as follows: GUUUUAGUACUCUG UAAUUUUA GAAA UAAAAUUA CAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAU (the underlined sequence is an additional 8 base pairs); the DNA sequence of the +8bp sgRNA backbone is shown in SEQ ID NO:7.

Example 2. Application of the modified SaCas9 sgRNA in improving the C·T base substitution efficiency of the SaKKHn&PmCDA1&UGI base editing system

1. Construction of recombinant expression vector

The following recombinant expression vectors were artificially synthesized, and each expression vector was a circular plasmid:

Two recombinant expression vectors containing Original sgRNA: SaKKHn-pBE-1 and SaKKHn-pBE-2;

Two recombinant expression vectors containing +3bp sgRNA: SaKKHn-pBE+3bp-1 and SaKKHn-pBE+3bp-2;

Two recombinant expression vectors containing +8bp sgRNA: SaKKHn-pBE+8bp-1 and SaKKHn-pBE+8bp-2.

The nucleotide sequence of the SaKKHn-pBE-1 recombinant expression vector is sequence 1 in the sequence listing. Among them, the 131-467th position of sequence 1 is the nucleotide sequence of the OsU3 promoter, the 474-550th and 648-724th positions are the nucleotide sequences of tRNA, and the 551-647th and 725th-821th are the nucleotide sequences of tRNA. The nucleotide sequences of the two sgRNAs targeting the OsWaxy gene are respectively, and the DNA sequences of the common sgRNA backbone (Original sgRNA backbone) of the two sgRNAs are positions 571-647 of sequence 1 or 745-821 of sequence 1 996-1286 is the nucleotide sequence of the OsU3 terminator; 1293-3006 of sequence 1 is the nucleotide sequence of the OsUbq3 promoter, and 3013-6225 is the coding sequence of the SaKKHn protein (without termination codon), encoding the SaKKHn protein shown in sequence 2; the 6511-7134th position of sequence 1 is the coding sequence of PmCDA1 protein (without stop codon), encoding the PmCDA1 protein shown in sequence 3; Position 7452 is the coding sequence of UGI protein, which encodes the UGI protein shown in sequence 4; position 7459-7653 of sequence 1 is the nucleotide sequence of the 35S terminator; position 7728-9720 of sequence 1 is the nucleus of the ZmUbi1 promoter The nucleotide sequence, the 9727-10749th position is the coding sequence of hygromycin phosphotransferase, and the 10779th-10994th position is the nucleotide sequence of CaMV35S polyA. The two targets in the SaKKHn-pBE-1 recombinant expression vector are T1 and T2 respectively, and the sequences are shown in Table 1.

The nucleotide sequence of the SaKKHn-pBE-2 recombinant expression vector is the sequence obtained by replacing the 474-995th position of the sequence 1 with the sequence 5, and keeping the other sequences unchanged. Among them, positions 1-77 and 175-251 of sequence 5 are nucleotide sequences of tRNA, and positions 78-174 and 252-348 are two targeting OsNRT1.1B gene and OsGRF4 gene, respectively. Nucleotide sequence of sgRNA. Positions 98-174 and 272-348 are the DNA sequences of the Original sgRNA backbone. The two targets in the SaKKHn-pBE-2 recombinant expression vector are T3 and T4, respectively, and the sequences are shown in Table 1.

The nucleotide sequence of the SaKKHn-pBE+3bp-1 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the sequence of the SaKKHn-pBE-1 recombinant expression vector with the DNA sequence of the +3bp sgRNA backbone shown in SEQ ID NO: 6, and The sequence obtained after keeping other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+3bp-2 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the sequence of the SaKKHn-pBE-2 recombinant expression vector with the DNA sequence of the +3bp sgRNA backbone shown in SEQ ID NO: 6, and The sequence obtained after keeping other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+8bp-1 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the sequence of the SaKKHn-pBE-1 recombinant expression vector with the DNA sequence of the +8bp sgRNA backbone shown in sequence 7, and The sequence obtained after keeping other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+8bp-2 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the sequence of the SaKKHn-pBE-2 recombinant expression vector with the DNA sequence of the +8bp sgRNA backbone shown in sequence 7, and The sequence obtained after keeping other sequences unchanged.

The target nucleotide sequences of each vector and the corresponding PAM sequences are shown in Table 1.

Table 1

Second, the acquisition of rice positive T0 seedlings

The SaKKHn-pBE-1 vector, SaKKHn-pBE-2 vector, SaKKHn-pBE+3bp-1 vector, SaKKHn-pBE+3bp-2 vector, SaKKHn-pBE+8bp-1 vector and SaKKHn-pBE+ The 8bp-2 vector is operated according to the following steps 1-9:

1. The vector was introduced into Agrobacterium EHA105 (product of Shanghai Weidi Biotechnology Co., Ltd., CAT#: AC1010) to obtain recombinant Agrobacterium.

2. Use medium (YEP medium containing 50 μg/ml kanamycin and 25 μg/ml rifampicin) to cultivate recombinant Agrobacterium, 28 ° C, 150 rpm shaking culture to OD ₆₀₀ of 1.0-2.0, at room temperature, 10000 rpm Centrifuge for 1 min, resuspend the cells with the infection solution (replace the sugar in the N6 liquid medium with glucose and sucrose, the concentrations of glucose and sucrose in the infection solution are 10 g/L and 20 g/L, respectively) and dilute to OD ₆₀₀ is 0.2 to obtain Agrobacterium infection solution.

3. The mature seeds of the rice variety Nipponbare were peeled and threshed, placed in a 100mL conical flask, soaked in 70% (v/v) ethanol aqueous solution for 30sec, then placed in 25% (v/v) sodium hypochlorite aqueous solution, sterilized by shaking at 120rpm for 30min , rinsed with sterile water for 3 times, blotted the water with filter paper, then placed the seed embryos face down on N6 solid medium, and cultivated in the dark at 28°C for 4-6 weeks to obtain rice callus.

4, after completing step 3, the rice callus is soaked and placed in Agrobacterium infection solution A (Agrobacterium infection solution A is the liquid obtained by adding acetosyringone to the Agrobacterium infection solution, and the addition of acetosyringone satisfies The volume ratio of acetosyringone and Agrobacterium infection solution is 25μl: 50ml) for 10min, and then placed on a petri dish (containing about 200ml of Agrobacterium-free infection solution) covered with two layers of sterile filter paper. , 21 ℃ dark culture for 1 day.

5. Take the rice callus obtained in step 4, put it on recovery medium, and cultivate in the dark at 25-28°C for 3 days.

6. Take the rice callus obtained in step 5, place it on the screening medium, and cultivate in the dark at 28°C for 2 weeks.

7. Take the rice callus obtained in step 6, place it on the screening medium again, and cultivate in the dark at 28° C. for 2 weeks to obtain the rice callus with resistance.

8. Take the rice resistant callus obtained in step 7 and put it on the differentiation medium, cultivate it in the light of 25°C for about 1 month, move the differentiated seedlings to the rooting medium, and cultivate in the light of 25°C for 2 weeks to obtain the rice T0 Seedling.

9. Extract the genomic DNA of rice T0 seedlings and use it as a template, use primers F (5'-attatgtagcttgtgcgtttcg-3') and primer R (5'-ctccacctcattgacattatgc-3') to form primer pairs to carry out PCR amplification to obtain PCR Amplification product; carry out agarose gel electrophoresis on this PCR amplification product, and then judge as follows: if the PCR amplification product contains a DNA fragment of about 898bp, the corresponding rice T0 seedling is a rice positive T0 seedling; If the amplified product does not contain a DNA fragment of about 898 bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.

3. Analysis of results

1. Take the genomic DNA of the rice-positive T0 seedlings obtained in step 2 as a template for each vector. For the T1 target, use the primer pair T1 to carry out PCR amplification to obtain a PCR amplification product; for the T2 target, use the primer pair T2 Perform PCR amplification to obtain PCR amplification products; for T3 targets, use primers to perform PCR amplification on T3 to obtain PCR amplification products; for T4 targets, use primers to perform PCR amplification on T4 to obtain PCR amplification products .

2. Sanger sequencing and analysis of the PCR amplification product obtained in step 1. Sequencing results were only analyzed for each target region. The number of positive T0 seedlings with C·T base substitution in T1, T2, T3 and T4 were counted respectively, and the C·T base substitution efficiency was calculated. The results are shown in Table 2.

The results showed that for all four targets, the SaKKHn&PmCDA1&UGI base editing system using +3bp sgRNA could improve the C T base substitution efficiency compared with the SaKKHn&PmCDA1&UGI base editing system using the Original sgRNA, but only for the T2 target. language, an increase of 3 times. The SaKKHn&PmCDA1&UGI base editing system using +8bp sgRNA is unstable, and it can improve the C·T base substitution efficiency for T2, T3 and T4 targets to varying degrees, but it reduces C to a certain extent for T1 targets. • T base substitution efficiency. On the overall synergistic level, except for the T4 target, the base editing system of SaKKHn&PmCDA1&UGI with +3bp sgRNA is more efficient than the base editing system of SaKKHn&PmCDA1&UGI with +8bp sgRNA in realizing C·T base editing.

Table 2

Example 3. Application of +3bp sgRNA in improving the C·T base substitution efficiency of SaKKHn&PmCDA1&UGI base editing system

1. Construction of recombinant expression vector

The following recombinant expression vectors were artificially synthesized, and each expression vector was a circular plasmid:

Five recombinant expression vectors containing Original sgRNA: SaKKHn-pBE-3, SaKKHn-pBE-4, SaKKHn-pBE-5, SaKKHn-pBE-6 and SaKKHn-pBE-7;

Five recombinant expression vectors containing +3bp sgRNA: SaKKHn-pBE+3bp-3, SaKKHn-pBE+3bp-4, SaKKHn-pBE+3bp-5, SaKKHn-pBE+3bp-6 and SaKKHn-pBE+3bp-7 .

The nucleotide sequence of the SaKKHn-pBE-3 recombinant expression vector is the sequence obtained by replacing the 474-995th position of the sequence 1 with the sequence 8, and keeping the other sequences unchanged. Wherein, the 1-77th position of sequence 8 is the nucleotide sequence of tRNA, the 78th-174th position is the nucleotide sequence of the sgRNA targeting the OsWaxy gene, and the 98th-174th position is the DNA sequence of the Original sgRNA backbone. The target in the SaKKHn-pBE-3 recombinant expression vector is T5, and the sequence is shown in Table 3.

The nucleotide sequence of the SaKKHn-pBE-4 recombinant expression vector is the sequence obtained by replacing the T5 target sequence in the SaKKHn-pBE-3 recombinant expression vector sequence with the T6 target sequence, and keeping other sequences unchanged. The T6 target sequences are shown in Table 3.

The nucleotide sequence of the SaKKHn-pBE-5 recombinant expression vector is the sequence obtained by replacing the T5 target sequence in the SaKKHn-pBE-3 recombinant expression vector sequence with the T7 target sequence, and keeping other sequences unchanged. The T7 target sequences are shown in Table 3.

The nucleotide sequence of the SaKKHn-pBE-6 recombinant expression vector is the sequence obtained by replacing the T5 target sequence in the SaKKHn-pBE-3 recombinant expression vector sequence with the T8 target sequence, and keeping other sequences unchanged. The T8 target sequence is shown in Table 3.

The nucleotide sequence of the SaKKHn-pBE-7 recombinant expression vector is the sequence obtained by replacing the T5 target sequence of the sequence in the SaKKHn-pBE-3 recombinant expression vector with the T9 target sequence, and keeping other sequences unchanged. The T9 target sequence is shown in Table 3.

The nucleotide sequence of the SaKKHn-pBE+3bp-3 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the sequence of the SaKKHn-pBE-3 recombinant expression vector with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6, and keep The sequence obtained with other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+3bp-4 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the sequence of the SaKKHn-pBE-4 recombinant expression vector with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6, and keep The sequence obtained with other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+3bp-5 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the sequence of the SaKKHn-pBE-5 recombinant expression vector with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6, and keep The sequence obtained with other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+3bp-6 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the sequence of the SaKKHn-pBE-6 recombinant expression vector with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6, and keep The sequence obtained with other sequences unchanged.

The nucleotide sequence of the SaKKHn-pBE+3bp-7 recombinant expression vector is to replace the DNA sequence of the Original sgRNA backbone in the sequence of the SaKKHn-pBE-7 recombinant expression vector with the DNA sequence of the +3bp sgRNA backbone shown in sequence 6, and keep The sequence obtained with other sequences unchanged.

The target nucleotide sequences of each vector and the corresponding PAM sequences are shown in Table 3.

table 3

target name target gene Target sequence (5'-3') PAM Recombinant expression vector name T5 OsWaxy tcctcggcgtagtacgggct CACGGT SaKKHn-pBE-3; SaKKHn-pBE+3bp-3 T6 OsWaxy tatccgggcaaggtgagggc CGTGGT SaKKHn-pBE-4; SaKKHn-pBE+3bp-4 T7 OsGRF4 acgccggcaccgccctggct CTGGGT SaKKHn-pBE-5; SaKKHn-pBE+3bp-5 T8 OsALS cccaagcatgcgcagggaca ACGGGT SaKKHn-pBE-6; SaKKHn-pBE+3bp-6 T9 OsALS cacgtccttcccgctcgagg CCGGGT SaKKHn-pBE-7; SaKKHn-pBE+3bp-7

Second, the acquisition of rice positive T0 seedlings

The SaKKHn-pBE-3 vector, SaKKHn-pBE-4 vector, SaKKHn-pBE-5 vector, SaKKHn-pBE-6 vector, SaKKHn-pBE-7 vector, SaKKHn-pBE+3bp-3 vector, SaKKHn-pBE-7 vector constructed in step 1, SaKKHn -pBE+3bp-4 vector, SaKKHn-pBE+3bp-5 vector, SaKKHn-pBE+3bp-6 vector and SaKKHn-pBE+3bp-7 vector were respectively operated according to 1-9 of step 2 in Example 2, to obtain Rice-positive T0 seedlings.

3. Analysis of results

1. Take the genomic DNA of the rice-positive T0 seedlings obtained in step 2 as a template for each vector. For the T5 target, use primer pair T5 to carry out PCR amplification to obtain a PCR amplification product; for the T6 target, use primer pair T6 Perform PCR amplification to obtain PCR amplification products; for T7 targets, use primers to perform PCR amplification on T4 to obtain PCR amplification products; for T8 targets, use primers to perform PCR amplification on T8 to obtain PCR amplification products ; For the T9 target, use primers to carry out PCR amplification on T9 to obtain PCR amplification products.

2. Sanger sequencing and analysis of the PCR amplification product obtained in step 1. Sequencing results were only analyzed for each target region. The number of positive T0 seedlings with C·T base substitution in T5, T6, T7, T8 and T9 were counted respectively, and the C·T base substitution efficiency was calculated. The results are shown in Table 4.

The results showed that for all five targets, the SaKKHn&PmCDA1&UGI base editing system using +3bp sgRNA could improve the C·T base substitution efficiency compared to the SaKKHn&PmCDA1&UGI base editing system using the Original sgRNA. For the T9 target only, the SaKKHn&PmCDA1&UGI base editing system using Original sgRNA cannot achieve C·T base substitution, while the SaKKHn&PmCDA1&UGI base editing system using +3bp sgRNA can successfully achieve C·T base editing.

Table 4

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

sequence listing

<110> Beijing Academy of Agriculture and Forestry

<120> An efficient sgRNA and its application in gene editing

<160>10

<170>PatentIn version 3.5

<210>1

<211>17400

<212> DNA

<213>Artificial Sequence

<400>1

ggtggcagga tatattgtgg tgtaaacatg gcactagcct caccgtcttc gcagacgagg 60

ccgctaagtc gcagctacgc tctcaacggc actgactagg tagtttaaac gtgcacttaa 120

ttaaggtacc gaagcaactt aaagttatca ggcatgcatg gatcttggag gaatcagatg 180

tgcagtcagg gaccatagca caagacaggc gtcttctact ggtgctacca gcaaatgctg 240

gaagccggga acactgggta cgttggaaac cacgtgatgt gaagaagtaa gataaactgt 300

aggagaaaag catttcgtag tgggccatga agcctttcag gacatgtatt gcagtatggg 360

ccggcccatt acgcaattgg acgacaacaa agactagtat tagtaccacc tcggctatcc 420

acatagatca aagctgattt aaaagagttg tgcagatgat ccgtggcgga tccaacaaag 480

caccagtggt ctagtggtag aatagtaccc tgccacggta cagacccggg ttcgattccc 540

ggctggtgca catccaatgc gatgatcaag gttttagtac tctggaaaca gaatctacta 600

aaacaaggca aaatgccgtg tttatctcgt caacttgttg gcgagataac aaagcaccag 660

tggtctagtg gtagaatagt accctgccac ggtacagacc cgggttcgat tcccggctgg 720

tgcaaatcac cagtggaagc taaggtttta gtactctgga aacagaatct actaaaacaa 780

ggcaaaatgc cgtgtttatc tcgtcaactt gttggcgaga taacaaagca ccagtggtct 840

agtggtagaa tagtaccctg ccacggtaca gacccgggtt cgattcccgg ctggtgcaac 900

cggatttgaa cgatggacgt tttagtactc tggaaacaga atctactaaa acaaggcaaa 960

atgccgtgtt tatctcgtca acttgttggc gagatttttt tttttcgttt tgcattgagt 1020

tttctccgtc gcatgtttgc agtttttattt tccgttttgc attgaaattt ctccgtctca 1080

tgtttgcagc gtgttcaaaa agtacgcagc tgtatttcac ttatttacgg cgccacattt 1140

tcatgccgtt tgtgccaact atcccgagct agtgaataca gcttggcttc acacaacact 1200

ggtgacccgc tgacctgctc gtacctcgta ccgtcgtacg gcacagcatt tggaattaaa 1260

gggtgtgatc gatactgctt gctgctaagc ttacaaattc gggtcaaggc ggaagccagc 1320

gcgccacccc acgtcagcaa atacggaggc gcggggttga cggcgtcacc cggtcctaac 1380

ggcgaccaac aaaccagcca gaagaaatta cagtaaaaaa aaagtaaatt gcactttgat 1440

ccacctttta ttacctaagt ctcaatttgg atcaccctta aacctatctt ttcaatttgg 1500

gccgggttgt ggtttggact accatgaaca acttttcgtc atgtctaact tccctttcag 1560

caaacatatg aaccatatat agaggagatc ggccgtatac tagagctgat gtgtttaagg 1620

tcgttgattg cacgagaaaa aaaaatccaa atcgcaacaa tagcaaattt atctggttca 1680

aagtgaaaag atatgtttaa aggtagtcca aagtaaaact tatagataat aaaatgtggt 1740

ccaaagcgta attcactcaa aaaaaatcaa cgagacgtgt accaaacgga gacaaacggc 1800

atcttctcga aatttcccaa ccgctcgctc gcccgcctcg tcttcccgga aaccgcggtg 1860

gtttcagcgt ggcggattct ccaagcagac ggagacgtca cggcacggga ctcctcccac 1920

cacccaaccg ccataaatac cagccccctc atctcctctc ctcgcatcag ctccaccccc 1980

gaaaaatttc tccccaatct cgcgaggctc tcgtcgtcga atcgaatcct ctcgcgtcct 2040

caaggtacgc tgcttctcct ctcctcgctt cgtttcgatt cgatttcgga cgggtgaggt 2100

tgttttgttg ctagatccga ttggtggtta gggttgtcga tgtgattatc gtgagatgtt 2160

taggggttgt agatctgatg gttgtgattt gggcacggtt ggttcgatag gtggaatcgt 2220

ggttaggttt tgggattgga tgttggttct gatgattggg gggaattttt acggttagat 2280

gaattgttgg atgattcgat tggggaaatc ggtgtagatc tgttggggaa ttgtggaact 2340

agtcatgcct gagtgattgg tgcgatttgt agcgtgttcc atcttgtagg ccttgttgcg 2400

agcatgttca gatctactgt tccgctcttg attgagttat tggtgccatg ggttggtgca 2460

aacacaggct ttaatatgtt atatctgttt tgtgtttgat gtagatctgt agggtagttc 2520

ttcttagaca tggttcaatt atgtagcttg tgcgtttcga tttgatttca tatgttcaca 2580

gattagataa tgatgaactc ttttaattaa ttgtcaatgg taaataggaa gtcttgtcgc 2640

tatatctgtc ataatgatct catgttacta tctgccagta atttatgcta agaactatat 2700

tagaatatca tgttacaatc tgtagtaata tcatgttaca atctgtagtt catctatata 2760

atctattgtg gtaatttctt tttactatct gtgtgaagat tattgccact agttcattct 2820

acttatttct gaagttcagg atacgtgtgc tgttactacc tatctgaata catgtgtgat 2880

gtgcctgtta ctatcttttt gaatacatgt atgttctgtt ggaatatgtt tgctgtttga 2940

tccgttgttg tgtccttaat cttgtgctag ttcttaccct atctgtttgg tgattatttc 3000

ttgcagtacg taatggctcc taagaagaag cggaaggttg gcatccacgg tgtcccggcg 3060

gcaaagagaa actacatcct gggtctggcc atcggtatta catcggtggg ctacggcatc 3120

atcgactacg agacaaggga tgtcatcgat gccggcgtcc ggctcttcaa ggaggccaac 3180

gtggagaata acgagggcag gcgctccaag cgcggcgcgc ggaggctgaa gcgcaggcgg 3240

aggcatcgca tccagcgggt gaagaagctc ctcttcgact acaatctgct cacggatcat 3300

tccgagctgt ctggcatcaa cccatacgag gcgcgggtga agggcctgtc ccagaagctc 3360

tcggaggagg agttctcggc ggccctgctg catctcgcga agaggcgcgg cgtgcataat 3420

gtcaatgagg tggaggagga taccggcaat gagctgtcaa ccaaggagca gatcagcagg 3480

aactccaagg cgctggagga gaagtatgtg gcggagctcc agctcgagag gctgaagaag 3540

gatggcgagg tccggggctc catcaatagg ttcaagacat cggactacgt gaaggaggcc 3600

aagcagctcc tgaaggtgca gaaggcgtac caccagctgg accagagctt catcgacacc 3660

tacatcgatc tgctcgagac acgccggacg tactacgagg gcccgggcga gggctcaccg 3720

ttcggctgga aggacatcaa ggagtggtac gagatgctga tgggccactg cacctacttc 3780

cctgaggagc tgaggagcgt gaagtacgcg tacaatgcgg acctctacaa cgccctgaac 3840

gacctcaata acctcgtgat cacgcgcgac gagaatgaga agctcgagta ctacgagaag 3900

ttccagatca tcgagaacgt gttcaagcag aagaagaagc cgaccctcaa gcagatcgcc 3960

aaggagatcc tcgtcaatga ggaggacatc aagggctaca gggtgacctc gaccggcaag 4020

ccagagttca ccaacctgaa ggtctaccac gacatcaagg atatcaccgc ccgcaaggag 4080

atcatcgaga atgcggagct cctggatcag atcgcgaaga tcctcaccat ctaccagtcc 4140

agcgaggaca tccaggagga gctcacgaac ctgaatagcg agctgaccca ggaggagatc 4200

gagcagatct ccaacctcaa gggctacacc ggcacgcaca atctgagcct caaggcgatc 4260

aatctcatcc tcgatgagct ctggcataca aatgataacc agatcgccat cttcaatcgc 4320

ctcaagctgg tcccaaagaa ggtcgatctg tcgcagcaga aggagatccc aacgacactg 4380

gtcgatgact tcatcctctc acctgtcgtg aagaggtcgt tcatccagtc gatcaaggtc 4440

atcaatgcga tcatcaagaa gtacggcctc cctaatgata tcatcatcga gctggcccgc 4500

gagaagaatt caaaggacgc gcagaagatg atcaacgaga tgcagaagag gaatcggcag 4560

acaaacgagc gcatcgagga gatcatccgc acaaccggca aggagaatgc caagtacctg 4620

atcgagaaga tcaagctgca tgacatgcag gagggcaagt gcctctactc actggaggcc 4680

atcccactcg aggacctgct gaataaccca ttcaattacg aggtcgacca tatcatcccg 4740

cgctccgtgt cgttcgacaa ttccttcaat aacaaggtcc tcgtcaagca ggaggagaac 4800

tccaagaagg gcaatcgcac cccgttccag tacctgtcct cttcggacag caagatctct 4860

tacgagacat tcaagaagca catcctcaac ctggccaagg gcaagggccg gatctccaag 4920

accaagaagg agtacctcct ggaggagagg gatatcaacc ggttcagcgt gcagaaggac 4980

ttcatcaatc gcaacctggt cgatacccgg tacgccacca ggggcctcat gaacctgctc 5040

cggtcctact tccgggtgaa caatctcgac gtgaaggtca agagcatcaa cggcggcttc 5100

acctcgttcc tcaggcggaa gtggaagttc aagaaggagc ggaacaaggg ctacaagcac 5160

catgccgagg acgccctcat catcgcgaac gcggacttca tcttcaagga gtggaagaag 5220

ctcgataagg cgaagaaggt catggagaac cagatgttcg aggagaagca ggccgagtcg 5280

atgccagaga tcgagacaga gcaggagtac aaggagatct tcatcacccc gcaccagatc 5340

aagcacatca aggacttcaa ggactacaag tactcccatc gggtcgataa gaagccaaat 5400

cggaagctca tcaatgatac cctctactcg acacgcaagg atgacaaggg caacaccctg 5460

atcgtcaata acctcaatgg cctctacgac aaggataacg acaagctgaa gaagctcatc 5520

aacaagagcc cagagaagct cctcatgtac caccacgatc cgcagacata ccagaagctc 5580

aagctgatca tggagcagta cggcgacgag aagaacccac tctacaagta ctacgaggag 5640

acaggcaact acctgaccaa gtactccaag aaggacaatg gcccagtgat caagaagatc 5700

aagtactacg gcaataagct gaacgcccac ctcgatatca cggacgatta ccctaacagc 5760

cggaataagg tggtcaagct gtccctcaag ccgtaccgct tcgacgtcta cctggataac 5820

ggcgtctaca agttcgtgac agtcaagaat ctcgacgtca tcaagaagga gaactactac 5880

gaggtcaatt ctaagtgcta cgaggaggcc aagaagctca agaagatcag caaccaggcc 5940

gagttcatcg ccagcttcta caagaacgat ctgatcaaga tcaacggcga gctctacagg 6000

gtcatcggcg tgaacaatga cctgctcaat aggatcgagg tgaacatgat cgacatcacc 6060

taccgcgagt acctcgagaa catgaacgat aagcggcctc cacacatcat caagacaatc 6120

gcctctaaga cccagtccat caagaagtac tccacggata tcctcggcaa cctctacgag 6180

gtgaagtcaa agaagcaccc gcagatcatc aagaagggct cggctggagg aggaggcacg 6240

ggaggaggag gctccgccga gtatgtgcgc gcgctcttcg acttcaacgg caatgacgag 6300

gaggatctcc ctttcaagaa gggcgacatc ctccgcatcc gcgataagcc ggaggagcag 6360

tggtggaacg cagaggactc cgagggcaag cggggcatga tcctggtgcc atacgtcgag 6420

aagtacagcg gcgattacaa ggaccacgat ggcgactaca aggatcatga catcgattac 6480

aaggacgatg acgataagtc cggcgtcgac atgacggacg cggagtatgt gcgcatccac 6540

gagaagctcg atatctacac cttcaagaag cagttcttca acaataagaa gtcggtgtcc 6600

catcggtgct acgtcctctt cgagctgaag cgcaggggag agcgccgcgc ctgcttctgg 6660

ggctacgcgg tgaataagcc gcagtcaggc acagagcgcg gcatccacgc cgagatcttc 6720

tcgatccgga aggtcgagga gtacctccgc gacaacccag gccagttcac gatcaattgg 6780

tactccagct ggtccccttg cgcagattgc gcagagaaga tcctcgagtg gtacaaccag 6840

gagctgaggg gcaatggcca taccctcaag atctgggcct gcaagctgta ctacgagaag 6900

aacgcgagga atcagatcgg cctctggaac ctgcgggata atggcgtggg cctcaacgtg 6960

atggtgtccg agcactacca gtgctgccgc aagatcttca tccagtcctc ccacaatcag 7020

ctgaacgaga ataggtggct cgaaaagacc ctgaagcgcg ccgagaagtg gaggagcgag 7080

ctgtctatca tgatccaggt caagatcctg cacaccacaa agtcaccggc ggtgggcggc 7140

ggcggcagcg aattctccgg cggcagcacg aacctcagcg acatcatcga gaaggagaca 7200

ggcaagcagc tcgtgatcca ggagtctatc ctcatgctgc ctgaggaggt ggaggaggtc 7260

atcggcaaca agccggagtc cgatatcctc gtgcacaccg cctacgacga gtcgacagat 7320

gagaatgtca tgctcctgac ctccgacgca ccagagtaca agccatgggc gctcgtgatc 7380

caggattcca acggcgagaa taagatcaag atgctgtctg gcggctcccc gaagaagaag 7440

cgcaaggtct agactagtct gaaatcacca gtctctctct acaaatctat ctctctctat 7500

aataatgtgt gagtagttcc cagataaggg aattagggtt cttatagggt ttcgctcatg 7560

tgttgagcat ataagaaacc cttagtatgt atttgtattt gtaaaatact tctatcaata 7620

aaatttctaa ttcctaaaac caaaatccag tggggcgccc gacctgtact cgcgaaggtt 7680

aacttacaga gagtgtccgg gcgcgcctgg tggatcgtcc gcctaggctg cagtgcagcg 7740

tgacccggtc gtgcccctct ctagagataa tgagcattgc atgtctaagt tataaaaaat 7800

taccacatat ttttttttgtc acacttgttt gaagtgcagt ttatctatct ttatacatat 7860

atttaaactt tactctacga ataatataat ctatagtact acaataatat cagtgtttta 7920

gagaatcata taaatgaaca gttagacatg gtctaaagga caattgagta ttttgacaac 7980

aggactctac agttttatct ttttagtgtg catgtgttct cctttttttt tgcaaatagc 8040

ttcacctata taatacttca tccattttat tagtacatcc atttagggtt tagggttaat 8100

ggtttttata gactaatttt tttagtacat ctattttatt ctattttagc ctctaaatta 8160

agaaaactaa aactctattt tagttttttt atttaataat ttagatataa aatagaataa 8220

aataaagtga ctaaaaatta aacaaatacc ctttaagaaa ttaaaaaaac taaggaaaca 8280

ttttttcttgt ttcgagtaga taatgccagc ctgttaaacg ccgtcgacga gtctaacgga 8340

caccaaccag cgaaccagca gcgtcgcgtc gggccaagcg aagcagacgg cacggcatct 8400

ctgtcgctgc ctctggaccc ctctcgagag ttccgctcca ccgttggact tgctccgctg 8460

tcggcatcca gaaattgcgt ggcggagcgg cagacgtgag ccggcacggc aggcggcctc 8520

ctcctcctct cacggcaccg gcagctacgg gggattcctt tcccaccgct ccttcgcttt 8580

cccttcctcg cccgccgtaa taaatagaca ccccctccac accctctttc cccaacctcg 8640

tgttgttcgg agcgcacaca cacacaacca gatctcccccc aaatccaccc gtcggcacct 8700

ccgcttcaag gtacgccgct cgtcctcccc ccccccccct ctctaccttc tctagatcgg 8760

cgttccggtc catggttagg gcccggtagt tctacttctg ttcatgtttg tgttagatcc 8820

gtgtttgtgt tagatccgtg ctgctagcgt tcgtacacgg atgcgacctg tacgtcagac 8880

acgttctgat tgctaacttg ccagtgtttc tctttgggga atcctgggat ggctctagcc 8940

gttccgcaga cgggatcgat ttcatgattt tttttgtttc gttgcatagg gtttggtttg 9000

cccttttcct ttatttcaat atatgccgtg cacttgtttg tcgggtcatc ttttcatgct 9060

ttttttttgtc ttggttgtga tgatgtggtc tggttgggcg gtcgttctag atcggagtag 9120

aattctgttt caaactacct ggtggattta ttaattttgg atctgtatgt gtgtgccata 9180

catattcata gttacgaatt gaagatgatg gatggaaata tcgatctagg ataggtatac 9240

atgttgatgc gggttttact gatgcatata cagagatgct ttttgttcgc ttggttgtga 9300

tgatgtggtg tggttgggcg gtcgttcatt cgttctagat cggagtagaa tactgtttca 9360

aactacctgg tgtatttatt aattttggaa ctgtatgtgt gtgtcataca tcttcatagt 9420

tacgagttta agatggatgg aaatatcgat ctaggatagg tatacatgtt gatgtgggtt 9480

ttactgatgc atatacatga tggcatatgc agcatctatt catatgctct aaccttgagt 9540

acctatctat tataataaac aagtatgttt tataattatt ttgatcttga tatacttgga 9600

tgatggcata tgcagcagct atatgtggat ttttttagcc ctgccttcat acgctattta 9660

tttgcttggt actgtttctt ttgtcgatgc tcaccctgtt gtttggtgtt acttctgcag 9720

gagctcatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag 9780

ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc 9840

ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac 9900

aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt 9960

gacattgggg agtttagcga gagcctgacc tattgcatct cccgccgttc acagggtgtc 10020

acgttgcaag acctgcctga aaccgaactg cccgctgttc tacaaccggt cgcggaggct 10080

atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg 10140

caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat 10200

gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc 10260

gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat 10320

ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc 10380

gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg 10440

ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga 10500

tcgccacgac tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg 10560

gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga 10620

tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc 10680

gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg 10740

gcaaagaaat agagtagatg ccgaccggga tctgtcgatc gacaagctcg agtttctcca 10800

taataatgtg tgagtagttc ccagataagg gaattagggt tcctataggg tttcgctcat 10860

gtgttgagca tataagaaac ccttagtatg tatttgtatt tgtaaaatac ttctatcaat 10920

aaaatttcta attcctaaaa ccaaaatcca gtactaaaat ccagatcccc cgaattaatt 10980

cggcgttaat tcagcctgca ggacgcgttt aattaagtgc acgcggccgc ctacttagtc 11040

aagagcctcg cacgcgactg tcacgcggcc aggatcgcct cgtgagcctc gcaatctgta 11100

cctagtgttt aaactatcag tgtttgacag gatatattgg cgggtaaacc taagagaaaa 11160

gagcgtttat tagaataacg gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca 11220

tttgtatgtg catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc 11280

ctccgctgct atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac 11340

atgtcgcaca agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt 11400

cttgtcgcgt gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac 11460

gccatgaaca agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac 11520

caggacttga ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc 11580

gagaagatca ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta 11640

cgccctggcg acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac 11700

ctactggaca ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag 11760

ccgtgggccg acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt 11820

gccgagttcg agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag 11880

gcccgaggcg tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc 11940

cgcgagctga tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg 12000

catcgctcga ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc 12060

aggcggcgcg gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc 12120

gagaatgaac gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt 12180

ttttcattac cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc 12240

ccgcgcacgt ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc 12300

tggcggcctg gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt 12360

gatgtgtatt tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag 12420

taaataaaca aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg 12480

cgggtcaggc aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc 12540

cgatgttctg ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg 12600

ggaagatcaa ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa 12660

ggccatcggc cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc 12720

tgtgtccgcg atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga 12780

catatgggcc accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg 12840

aaggctacaa gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga 12900

ggttgccgag gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg 12960

cgtgagctac ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg 13020

cgacgctgcc cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt 13080

taatgaggta aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc 13140

gcacgcagca gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg 13200

gtcaactttc agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa 13260

ggcaagacca ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc 13320

aaatgaataa atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga 13380

acaaccaggc accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg 13440

cgtaagcggc tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga 13500

atcggcgtga cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg 13560

acctggtgga gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag 13620

cacgccccgg tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac 13680

cgccggcagc cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt 13740

ttttcgttcc gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg 13800

ccgttttccg tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc 13860

cagacgggca cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg 13920

acctggtact gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga 13980

agggagacaa gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc 14040

ggcgagccga tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca 14100

ccacgcacgt tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat 14160

ccgagggtga agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg 14220

agtacatcga gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc 14280

cggacgtgct gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc 14340

tctaccgcct ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga 14400

tctacgaacg cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc 14460

tgatcgggtc aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc 14520

cgatcctagt catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat 14580

gtacggagca gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct 14640

ttcctgtgga tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt 14700

acattgggaa cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa 14760

aagagaaaaa aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa 14820

cccgcctggc ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc 14880

ctacccttcg gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg 14940

ctggccgctc aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg 15000

cgccgtcgcc actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt 15060

gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa 15120

gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg 15180

ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg 15240

catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg 15300

taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct 15360

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 15420

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 15480

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 15540

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 15600

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 15660

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 15720

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 15780

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 15840

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 15900

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 15960

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 16020

gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 16080

gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 16140

acgaaaactc acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca 16200

gtaaaatata atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata 16260

gctcgacata ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt 16320

cataccactt gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat 16380

ctttcacaaa gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg 16440

gcttttccgt ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt 16500

cccagttttc gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta 16560

agcggctgtc taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc 16620

tgatgcactc cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt 16680

ccgagcaaag gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt 16740

caaagtgcag gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt 16800

cccgttccac atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt 16860

tttcattttc tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta 16920

cgcagcggta tttttcgatc agttttttca attccggtga tattctcatt ttagccattt 16980

attatttcct tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa 17040

gacgaactcc aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt 17100

ttcaaagttg ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc 17160

gcggtgatca caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga 17220

gatcatccgt gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac 17280

atgagcaaag tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg 17340

ctgcctgtat cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct 17400

<210>2

<211>1071

<212> PRT

<213>Artificial Sequence

<400>2

Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala

1 5 10 15

Ala Lys Arg Asn Tyr Ile Leu Gly Leu Ala Ile Gly Ile Thr Ser Val

20 25 30

Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly

35 40 45

Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg

50 55 60

Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile

65 70 75 80

Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His

85 90 95

Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu

100 105 110

Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu

115 120 125

Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr

130 135 140

Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala

145 150 155 160

Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys

165 170 175

Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr

180 185 190

Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln

195 200 205

Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg

210 215 220

Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys

225 230 235 240

Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe

245 250 255

Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr

260 265 270

Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn

275 280 285

Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe

290 295 300

Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu

305 310 315 320

Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys

325 330 335

Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr

340 345 350

Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala

355 360 365

Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu

370 375 380

Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser

385 390 395 400

Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile

405 410 415

Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala

420 425 430

Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln

435 440 445

Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro

450 455 460

Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile

465 470 475 480

Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg

485 490 495

Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys

500 505 510

Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr

515 520 525

Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp

530 535 540

Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu

545 550 555 560

Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro

565 570 575

Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys

580 585 590

Gln Glu Glu Asn Ser Lys Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu

595 600 605

Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile

610 615 620

Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu

625 630 635 640

Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp

645 650 655

Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu

660 665 670

Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys

675 680 685

Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp

690 695 700

Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp

705 710 715 720

Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys

725 730 735

Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys

740 745 750

Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu

755 760 765

Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp

770 775 780

Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Lys Leu Ile

785 790 795 800

Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu

805 810 815

Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu

820 825 830

Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His

835 840 845

Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly

850 855 860

Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr

865 870 875 880

Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile

885 890 895

Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp

900 905 910

Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr

915 920 925

Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val

930 935 940

Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser

945 950 955 960

Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala

965 970 975

Glu Phe Ile Ala Ser Phe Tyr Lys Asn Asp Leu Ile Lys Ile Asn Gly

980 985 990

Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile

995 1000 1005

Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn

1010 1015 1020

Met Asn Asp Lys Arg Pro Pro His Ile Ile Lys Thr Ile Ala Ser

1025 1030 1035

Lys Thr Gln Ser Ile Lys Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn

1040 1045 1050

Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys

1055 1060 1065

Gly Ser Ala

1070

<210>3

<211>208

<212> PRT

<213>Artificial Sequence

<400>3

Met Thr Asp Ala Glu Tyr Val Arg Ile His Glu Lys Leu Asp Ile Tyr

1 5 10 15

Thr Phe Lys Lys Gln Phe Phe Asn Asn Lys Lys Ser Val Ser His Arg

20 25 30

Cys Tyr Val Leu Phe Glu Leu Lys Arg Arg Gly Glu Arg Arg Ala Cys

35 40 45

Phe Trp Gly Tyr Ala Val Asn Lys Pro Gln Ser Gly Thr Glu Arg Gly

50 55 60

Ile His Ala Glu Ile Phe Ser Ile Arg Lys Val Glu Glu Tyr Leu Arg

65 70 75 80

Asp Asn Pro Gly Gln Phe Thr Ile Asn Trp Tyr Ser Ser Trp Ser Pro

85 90 95

Cys Ala Asp Cys Ala Glu Lys Ile Leu Glu Trp Tyr Asn Gln Glu Leu

100 105 110

Arg Gly Asn Gly His Thr Leu Lys Ile Trp Ala Cys Lys Leu Tyr Tyr

115 120 125

Glu Lys Asn Ala Arg Asn Gln Ile Gly Leu Trp Asn Leu Arg Asp Asn

130 135 140

Gly Val Gly Leu Asn Val Met Val Ser Glu His Tyr Gln Cys Cys Arg

145 150 155 160

Lys Ile Phe Ile Gln Ser Ser His Asn Gln Leu Asn Glu Asn Arg Trp

165 170 175

Leu Glu Lys Thr Leu Lys Arg Ala Glu Lys Trp Arg Ser Glu Leu Ser

180 185 190

Ile Met Ile Gln Val Lys Ile Leu His Thr Thr Lys Ser Pro Ala Val

195 200 205

<210>4

<211>98

<212> PRT

<213>Artificial Sequence

<400>4

Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly

1 5 10 15

Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val

20 25 30

Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr

35 40 45

Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp

50 55 60

Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly

65 70 75 80

Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg

85 90 95

Lys Val

<210>5

<211>522

<212> DNA

<213>Artificial Sequence

<400>5

aacaaagcac cagtggtcta gtggtagaat agtaccctgc cacggtacag acccgggttc 60

gattcccggc tggtgcagat gccacacagc aaggagtgtt ttagtactct ggaaacagaa 120

tctactaaaa caaggcaaaa tgccgtgttt atctcgtcaa cttgttggcg agataacaaa 180

gcaccagtgg tctagtggta gaatagtacc ctgccacggt acagacccgg gttcgattcc 240

cggctggtgc acagaaccga caacagatga ggttttagta ctctggaaac agaatctact 300

aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagataa caaagcacca 360

gtggtctagt ggtagaatag taccctgcca cggtacagac ccgggttcga ttcccggctg 420

gtgcaccagc tcatttggct cggcggtttt agtactctgg aaacagaatc tactaaaaca 480

aggcaaaatg ccgtgtttat ctcgtcaact tgttggcgag at 522

<210>6

<211>83

<212> DNA

<213>Artificial Sequence

<400>6

gttttagtac tctgctggaa acagcagaat ctactaaaac aaggcaaaat gccgtgttta 60

tctcgtcaac ttgttggcga gat 83

<210>7

<211>93

<212> DNA

<213>Artificial Sequence

<400>7

gttttagtac tctgtaattt tagaaataaa attacagaat ctactaaaac aaggcaaaat 60

gccgtgttta tctcgtcaac ttgttggcga gat 93

<210>8

<211>174

<212> DNA

<213>Artificial Sequence

<400>8

aacaaagcac cagtggtcta gtggtagaat agtaccctgc cacggtacag acccgggttc 60

gattcccggc tggtgcatcc tcggcgtagt acgggctgtt ttagtactct ggaaacagaa 120

tctactaaaa caaggcaaaa tgccgtgttt atctcgtcaa cttgttggcg agat 174

<210>9

<211>77

<212> RNA

<213>Artificial Sequence

<400>9

guuuuaguac ucuggaaaca gaaucuacua aaacaaggca aaaugccgug uuuaucucgu 60

caacuuguug gcgagau 77

<210>10

<211>83

<212> RNA

<213>Artificial Sequence

<400>10

guuuuaguac ucugcuggaa acagcagaau cuacuaaaac aaggcaaaau gccguguuua 60

ucucgucaac uuguuggcga gau 83

Claims (10)

1. A kit comprising a sgRNA or a biological material associated with the sgRNA, a Cas9 nuclease or a biological material associated with the Cas9 nuclease, a cytosine deaminase or a biological material associated with the cytosine deaminase;

the sgRNA targets a target sequence;

the sgRNA is shown as formula I: an RNA-engineered sgRNA backbone transcribed from the target sequence (formula I);

the modified sgRNA framework is an RNA molecule obtained by inserting an RNA fragment A between 14 th and 15 th positions of the sgRNA framework and inserting an RNA fragment B between 18 th and 19 th positions of the sgRNA framework;

the RNA segment A and the RNA segment B are reversely complementary;

the sizes of the RNA fragment A and the RNA fragment B are both 3 nt;

the sgRNA backbone is m1) or m2) or m 3):

m1) the RNA molecule shown as the sequence 9;

m2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in m1) and having the same function;

m3) and m1) or m2) and has the same function.

2. The kit of claim 1, wherein: the engineered sgRNA backbone is n1) or n2) or n 3):

n1) the RNA molecule shown as the sequence 10;

n2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in n1) and having the same function;

n3) and n1) or n2) and has the same function.

3. The kit of claim 1 or 2, wherein: the Cas9 nuclease is a SaKKHn protein;

the SaKKHn protein is E1) or E2) or E3):

E1) the amino acid sequence is a protein shown in a sequence 2;

E2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;

E3) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of E1) or E2);

the biological material related to the SaKKHn is any one of F1) to F5):

F1) a nucleic acid molecule encoding said SaKKHn protein;

F2) an expression cassette comprising the nucleic acid molecule of F1);

F3) a recombinant vector comprising the nucleic acid molecule of F1) or a recombinant vector comprising the expression cassette of F2);

F4) a recombinant microorganism containing F1) said nucleic acid molecule, or a recombinant microorganism containing F2) said expression cassette, or a recombinant microorganism containing F3) said recombinant vector;

F5) a transgenic cell line comprising the nucleic acid molecule of F1) or a transgenic cell line comprising the expression cassette of F2).

4. The kit of claim 1 or 2, wherein: the cytosine deaminase is PmCDA1 protein;

the PmCDA1 protein is G1) or G2) or G3):

G1) the amino acid sequence is a protein shown in a sequence 3;

G2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 3 in the sequence table and has the same function;

G3) a fusion protein obtained by connecting a tag to the N-terminus or/and the C-terminus of G1) or G2);

the biological material related to the PmCDA1 protein is any one of H1) to H5):

H1) a nucleic acid molecule encoding the PmCDA1 protein;

H2) an expression cassette comprising the nucleic acid molecule of H1);

H3) a recombinant vector containing H1) the nucleic acid molecule or a recombinant vector containing H2) the expression cassette;

H4) a recombinant microorganism containing H1) the nucleic acid molecule, or a recombinant microorganism containing H2) the expression cassette, or a recombinant microorganism containing H3) the recombinant vector;

H5) a transgenic cell line containing H1) the nucleic acid molecule or a transgenic cell line containing H2) the expression cassette.

5. The kit of any one of claims 1 to 4, wherein: the sgRNA is tRNA-sgRNA;

the tRNA-sgRNA is shown as a formula I: tRNA-the RNA transcribed from the target sequence-engineered sgRNA backbone (formula I);

the tRNA is 1) or 2) or 3):

1) an RNA molecule obtained by replacing T in the 474-550 th position of the sequence 1 with U;

2) RNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the RNA molecules shown in 1) and have the same functions;

3) RNA molecule with 75% or more than 75% identity with the nucleotide sequence defined in 1) or 2) and with the same function.

6. The kit of any one of claims 1 to 5, wherein: the kit further comprises a UGI protein or a biological material associated with the UGI protein;

the UGI protein is I1) or I2) or I3):

I1) the amino acid sequence is a protein shown in a sequence 4;

I2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 4 in the sequence table and has the same function;

I3) a fusion protein obtained by connecting labels at the N terminal or/and the C terminal of I1) or I2);

the biological material related to the UGI protein is any one of J1) to J5):

J1) a nucleic acid molecule encoding the UGI protein;

J2) an expression cassette comprising the nucleic acid molecule of J1);

J3) a recombinant vector comprising J1) said nucleic acid molecule, or a recombinant vector comprising J2) said expression cassette;

J4) a recombinant microorganism containing J1) the nucleic acid molecule, or a recombinant microorganism containing J2) the expression cassette, or a recombinant microorganism containing J3) the recombinant vector;

J5) a transgenic cell line comprising J1) the nucleic acid molecule or a transgenic cell line comprising J2) the expression cassette.

7. The sgRNA of any one of claims 1-6 or the engineered sgRNA backbone of any one of claims 1-6.

8. The kit of any one of claims 1-6, or the sgRNA of claim 7, or the modified sgRNA backbone of claim 7, for use in any one of X1) -X4):

x1) editing of a target sequence in the genome of an organism or cell of an organism;

x2) preparing an edited product of a target sequence of a genome of an organism or a cell of an organism;

x3) increasing the efficiency of editing a target sequence in the genome of an organism or cell of an organism;

x4) to produce a product that increases the efficiency of editing a target sequence in the genome of an organism or cell of an organism.

9, Y1) or Y2):

y1) or a method of increasing the efficiency of editing a genomic target sequence of an organism or a cell of an organism, comprising expressing the sgRNA of any one of claims 1 to 6, the Cas9 nuclease of any one of claims 1 to 6, the cytosine deaminase of any one of claims 1 to 6 in the organism or cell of the organism to effect editing of the genomic target sequence; the sgRNA targets the target sequence;

y2) biological mutant, comprising the following steps: editing the genome of the organism according to the method described in Y1) to obtain a biological mutant.

10. The kit of any one of claims 1 to 6 or the use of claim 8 or the method of claim 9, wherein:

editing the genome target sequence to mutate C in the target sequence into T;

and/or, the organism is S1) or S2) or S3) or S4):

s1) plants or animals;

s2) a monocot or dicot;

s3) gramineous plants;

s4) rice;

and/or, the biological cell is T1) or T2) or T3) or T4):

t1) plant cells or animal cells;

t2) a monocotyledonous or dicotyledonous plant cell;

t3) graminaceous plant cells;

t4) rice cells.

Images