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WO2023216764A1 - 可降低基因编辑脱靶率的基因编辑蛋白变体 - Google Patents

可降低基因编辑脱靶率的基因编辑蛋白变体 Download PDF

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WO2023216764A1
WO2023216764A1 PCT/CN2023/085720 CN2023085720W WO2023216764A1 WO 2023216764 A1 WO2023216764 A1 WO 2023216764A1 CN 2023085720 W CN2023085720 W CN 2023085720W WO 2023216764 A1 WO2023216764 A1 WO 2023216764A1
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gene editing
variant
protein
protein variant
editing protein
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PCT/CN2023/085720
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English (en)
French (fr)
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尹蕾
王金
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上海吐露港生物科技有限公司
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Publication of WO2023216764A1 publication Critical patent/WO2023216764A1/zh

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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
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    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to the field of biotechnology, and specifically to gene editing protein variants that can reduce the off-target rate of gene editing.
  • Gene editing refers to operations such as deleting, inserting or replacing DNA sequences, and is widely used in gene function research, disease model building, disease treatment, and transgenic animal and plant engineering, etc.
  • the first generation of gene editing technology is based on zinc finger nuclease (Zinc Finger Nuclease, ZFN).
  • ZFN contains a DNA zinc finger binding domain that can specifically recognize sequences. By modifying this region, it can target different DNA sequences.
  • a DNA zinc finger binding domain is generally composed of multiple zinc finger structures. Each zinc finger structure recognizes 3 bases, so the target sequence of ZNF must be a multiple of 3. Due to the context-dependent effect of the recognition domain of ZNF, its design and screening are very difficult, and its application scope is limited.
  • the second-generation gene editing technology is based on Transcription Activator-like effector Nuclease (TALEN).
  • TALEN Transcription Activator-like effector Nuclease
  • the unit module that recognizes the DNA specificity of the target site is a doublet of amino acids separated by 32 constant amino acid residues.
  • the doublet of amino acids can correspond one-to-one with the four nucleotide bases of AGTC.
  • the corresponding double amino acid sequence is deduced to form the TALEN target recognition module. Assembly of this module requires a large number of molecular cloning and sequencing operations, thus limiting the promotion of this technology.
  • the third gene editing technology is based on CRISPR-Cas technology, which achieves specific recognition of target DNA sequences through guide RNA.
  • the design and synthesis workload of guide RNA is much smaller than the construction process of the DNA recognition module of TALEN and ZFN technology.
  • Guide RNA can bind to Cas protein with nuclease activity and guide it to cleave target DNA.
  • gene editing proteins still have a certain degree of off-target rate.
  • a gene editing protein such as Cas12a
  • it not only has cis-cleaving activity on the target DNA, but also has non-specific trans-cleaving activity on the single-stranded DNA present in the system.
  • double-stranded DNA will unwind into single-stranded DNA.
  • the trans-cleaving activity of gene editing proteins may cause these DNAs to be cleaved, thus leading to off-target production and causing cytotoxicity problems. . Therefore, it is necessary to eliminate gene editing proteins (such as Cas12a) Trans-cleavage activity to resolve off-target cytotoxicity issues.
  • the purpose of the present invention is to provide a method for reducing or even eliminating the trans-cleaving activity of gene editing proteins (such as Cas12a) to solve the problem of cytotoxicity caused by off-target.
  • gene editing proteins such as Cas12a
  • a first aspect of the present invention provides a gene editing protein variant, the variant is a non-natural protein with cis-cleaving activity, and the trans-cleaving activity of the variant is reduced compared to its wild-type gene editing protein , and the variant is mutated at one or more core amino acid positions of the wild-type gene editing protein selected from the following group:
  • the trans-cleavage activity of the variant is reduced compared with its wild-type gene editing protein, which means that the trans-cleavage activity of the variant is reduced by ⁇ 50% compared with the wild-type gene editing protein. , preferably ⁇ 80%, more preferably, ⁇ 90% or 100%.
  • the phenylalanine (F) at position 1081 of FnCas12a is mutated to one or more amino acids selected from the following group: arginine (R), tyrosine (Y), tryptophan (W), glutamine (Q), asparagine (N), lysine (K), glutamic acid (E), aspartic acid (D) or combinations thereof.
  • lysine (K) at position 1069 of FnCas12a is mutated to one or more amino acids selected from the following group: arginine (R), tyrosine (Y), glutamine ( Q), asparagine (N), lysine (K), glutamic acid (E), aspartic acid (D) or combinations thereof.
  • phenylalanine (F) at position 1081 of FnCas12a is mutated to arginine (R).
  • lysine (K) at position 1069 of FnCas12a is mutated to arginine (R).
  • the mutation is selected from the following group: F1081R, K1069R, or a combination thereof.
  • the gene editing protein is a V-type CRISPR/Cas protein.
  • the gene editing protein is selected from the following group: Cas 12, Cas 14, or a combination thereof.
  • the gene editing protein is selected from the following group: Cas12a, Cas12b, Cas12e or a combination thereof.
  • the Cas12a is selected from the following group: FnCas12a, LbCas12a, ErCas12a, Evcas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, CeCas12a, PrCas12a, CsbCas12a, BhCas12a , SsCas12a, Lb3Cas12a, BpCas12a, PdCas12a, BfCas12a, PcCas12a, cMtCas12a, PeCas12a, LiCas12a, Lb2Cas12a, PmCas12a, MbCas12a, EeCas12a, CsbCas12a, ArCas12a
  • the source of Cas12a is selected from the following group: Ciliatomyces, Listeria, Corynebacterium, Sutterella, Legionella, Treponema, Geneticobacter, Eurycoma, Bacteria, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Azospirillum, Sphaerochaeta, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus spp., Nitratifractor, Mycoplasma spp., Campylobacter spp., Lachnospira spp., or combinations thereof.
  • the source of Cas12a is selected from the following group: Francisella tularensis (FnCas12a), Acidaminococcus sp. BV3L6 (AsCas12a), Lachnospiraceae bacteria ND2006 ( Lachnospiraceae bacterium ND2006) (LbCas12a), Lachnospiraceae bacterium NC2008 (Lb5Cas12a), Helcococcus sp kunzii (HkCas12a), Oribacterium sp.NK2B42 (OsCas12a), Thiomicrospira sp.XS5 (T sCas12a ), Bacteroidetes oral taxon KA00251 (BbCas12a), Bacteroidetes oral taxon 274 (BoCas12a), Lachnospiraceae bacterium MC2017 (Lb4Cas12a), Coprococcus eu
  • SC_K08D17 (SsCas12a), Lachnospiraceae bacterium MC2017(Lb3Cas12a), Bytyrivibrio proteoclasticus(BpCas12a), Prevotella disens(PdCas12a), Fibrinolyticin Butyrivibrio fibrisolvens MD2001 (BfCas12a), Porphyromonas crevioricanis PcCas12a, Candidatus Methanoplasma termitum (CMtCas12a), Peregrinibacteria bacterium (PeCas12a), Leptospira inadaiserovar Lyme (LiCas12a ), Lachnospiraceae bacterium MA2020 (Lb2Cas12a), Porphyromonas macaca) (PmCas12a), Moraxella bovoculi 237 (MbCas12a), Eubacterium eligens (
  • the source of Cas12b is selected from the following group: Alicyclobacillus kakegawensis, Bacillus species V3-13, Bacillus hisashii, Viscose Lentisphaeria bacterium, Laceyella sediminis, or combinations thereof.
  • the Cas12b is selected from the following group: AacCas12b, AaCas12b, BthCas12b, AapCas12b, AkCas12b, AmCas12b, Bs3Cas12b, LsCas12b or a combination thereof.
  • the source of the gene editing protein is selected from the following group: Ciliatomyces, Listeria, Corynebacterium, Sutterella, Legionella, Treponema, and Geneticobacter , Eubacteria, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Azospirillum, Sphaerochaeta, Gluconacetobacter, Neisseria, Rosebura, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, Campylobacter, Lachnospira, or combinations thereof.
  • the source of the gene editing protein is selected from the following group: Lachnospiraceae bacterium ND2006 (LbCas12a), Thiomicrospira sp.XS5 (TsCas12a), Francisella tularensis (FnCas12a), Bacteroidetes oral taxon 274 (BoCas12a), Oribacterium sp.NK2B42 (OsCas12a), Acidaminococcus sp.BV3L6 (AsCas12a), Helcococcus sp kunzii (HkCas12a), Lachnospira Lachnospiraceae bacterium NC2008 (Lb5Cas12a), or a combination thereof.
  • the sites at positions 1081 and 1069 of FnCas12a are located at positions 1081 and 1069 of FnCas12a.
  • the sites at positions 1081 and 1069 of FnCas12a are located at positions 1019 and 1007 of BbCas12a.
  • the sites at positions 1081 and 1069 of FnCas12a are located at positions 1069 and 1057 of AsCas12a.
  • the sites at positions 1081 and 1069 of FnCas12a are located at positions 1033 and 1021 of BoCas12a.
  • the sites at positions 1081 and 1069 of FnCas12a are located at positions 1090 and 1078 of HkCas12a.
  • the sites at positions 1081 and 1069 of FnCas12a are located at positions 1004 and 992 of Lb4Cas12a.
  • the sites at positions 1081 and 1069 of FnCas12a are located at positions 980 and 968 of Lb5Cas12a.
  • the sites at positions 1081 and 1069 of FnCas12a are located at positions 1018 and 1006 of LbCas12a.
  • the sites at positions 1081 and 1069 of FnCas12a are located at positions 1001 and 989 of OsCas12a.
  • the sites at positions 1081 and 1069 of FnCas12a are located at positions 1070 and 1058 of TsCas12a.
  • the gene editing protein is FnCas12a.
  • sequence of the gene editing protein is shown in SEQ ID NO. 1.
  • amino acid sequence of the variant is as shown in any one of SEQ ID NO. 2-3.
  • the variant is a polypeptide having the amino acid sequence shown in any one of SEQ ID NO.: 2-3, its active fragment, or its conservative variant polypeptide.
  • the remaining amino acid sequences of the variant are identical or substantially identical to the sequence of the wild-type gene editing protein.
  • the basic similarity means that at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acids are different, wherein, The differences include amino acid substitutions, deletions or additions, and the variants have cis-cleaving activity and reduced trans-cleaving activity.
  • the homology of the variant and the wild-type gene editing protein is at least 80%, preferably at least 85% or 90%, more preferably at least 95%, and optimally Land is at least 98% or 99%.
  • the variant is selected from the following group:
  • amino acid sequence shown in any one of SEQ ID NO.: 2-3 is formed by the substitution, deletion or addition of one or more (such as 2, 3, 4 or 5) amino acid residues , and a polypeptide derived from (a) that has cis-cleaving activity and reduced trans-cleaving activity.
  • the derived polypeptide is identical to any of the sequences shown in SEQ ID NO.: 2-3.
  • the origin is at least 60%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%, such as 95%, 97%, 99%.
  • the variant is formed by mutation of the wild-type gene editing protein.
  • a second aspect of the invention provides a polynucleotide encoding the variant described in the first aspect of the invention.
  • polynucleotide is selected from the following group:
  • the polynucleotide additionally contains auxiliary elements selected from the following group on the flanks of the ORF of the variant: signal peptide, secreted peptide, tag sequence (such as 6His), or a combination thereof.
  • the polynucleotide is selected from the group consisting of genomic sequences, cDNA sequences, RNA sequences, or combinations thereof.
  • the polynucleotide further includes a promoter operably linked to the ORF sequence of the variant.
  • the promoter is selected from the following group: a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a strong promoter.
  • a third aspect of the present invention provides a vector, said vector containing the polynucleotide described in the second aspect of the present invention.
  • the vector includes one or more promoters operably linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, replication origin, and selectable marker. , nucleic acid restriction sites, and/or homologous recombination sites.
  • the vector includes a plasmid vector, a phage vector, a cosmid cloning vector, a phagemid vector, an artificial chromosome vector, an episomal vector, a viral vector or a combination thereof.
  • the artificial chromosome vector is selected from the following group: bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), P1 artificial chromosome (PAC) or a combination thereof;
  • the viral vector is selected from the following group: retroviral vectors, adenoviral vectors, Adeno-associated virus vector, herpesvirus vector, poxvirus vector, baculovirus vector, papillomavirus vector, papillomavirus vector, HBP Epstein Barrvirus vector, vaccinia virus vector, Semliki Forest Virus (Semliki Forest virus; SFV) or combination thereof;
  • the vector is selected from the following group: pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, pBJ, pGEX, VSV, pBR322, pCMV-HA, pEN, YAC, BAC, lambda phage, M13 phage, phagemid , pCAS9, pCEN6, pYES1L, p3HPRT1, pFN2A, pBC, pTZ, pGEM, pGEMK, pEX, pSAR, pCEP, cosmid, pBluescript, pKJK, pFloxin, pCP, pHR, pUC, pMAL, pALTER, pBAD, pCal, pL , pET, pGEMEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, p
  • the vector includes a cloning vector, a transformation vector, an expression vector, a shuttle vector, an integration vector, and a multifunctional vector.
  • the fourth aspect of the present invention provides a host cell, which contains the vector described in the third aspect of the present invention, or has the polynucleotide described in the second aspect of the present invention integrated into its genome.
  • the host cell is a prokaryotic recipient cell.
  • the prokaryotic receptor cells are selected from the following group: Escherichia coli, lactic acid bacteria, Bacillus subtilis, cyanobacteria, Streptomyces, Pseudomonas, Propionibacterium, Pectinatus sp., Bacteroides sp., Bacillus subtilis, Streptomyces, Anabaena, Arthrobacter, Agrobacterium Agrobacterium, Acetobacter, Acetobacterium, Bacillus, Brevibacillus, Bifidobacterium, Brachybacterium , Brevibacterium, Carnobacterium, Xenorhabdus, Photorhabdus, Corynebacterium, Enterobacter, Pasteurella ), Lactobacillus, Alcaligenes, Flavobacterium spp., Clostridium, Pasteuria, Escherichia ( Escherichia), Gluconacetobacter, Gluconobacter, Hafnia, Halomon
  • the prokaryotic receptor cell is selected from the following group: Escherichia Coli., Rodhobacter sphaeroides, Pseudoalteromonas haloplanktis, Shewanella Shewanella sp.
  • strain Ac10 Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aeruginosa, P.alcaligenes ), Pseudomonas aeruginosa PAO1-LAC, Pseudomonas putida KT2440, Halomonas elongata, Pseudoalteromonas citrea, Chromohalobacter salex'igens ), Streptomyces lividans, Streptomyces griseus, Streptomyces coelicolor, S.avermitilis, Streptomyces griseus, Streptomyces scabies (S.scabies), Streptomyces lividans (S.lividans) TK24, light Streptomyces lividans 1326, Nocardia lactamdurans, Mycobacterium smegmatis, Coryne
  • the E. coli is selected from the following group: BL21, BL21 (DE3), W3110, MG1655, RB791, RV308, HMS 174, HMS174 (DE3), NM533, XL1-Blue, C600, DH1, HB101, JM109, Top10, DH5 ⁇ , DH10 ⁇ , TG1, BW23473, BW23474, MW003, MW005 cells or combinations thereof; the Bacillus megaterium (Bacillus megaterium) is selected from the following group: QMB1551, PV361, DSM319 or combinations thereof.
  • the host cell is a eukaryotic cell.
  • the eukaryotic receptor cells are selected from the following group: yeast, fungi, plant cells, animal cells or combinations thereof.
  • the yeast is selected from the following species: Rhodotorula spp., Aureobasidium spp., Saccharomyces spp., Saccharomyces spp. Species (Sporobolomyces spp.), and their combinations.
  • the yeast is selected from the following group: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis Yeast (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces weldenii (ATCC 24,178), Kluyveromyces vartiensis (ATCC 56,500), Kluyveromyces vartiensis (ATCC 56,500) K.waltii) (ATCC 56500), Kluyveromyces drosophila (ATCC 36,906), Kluyveromyces thermotolerans, Kluyveromyces marxianus, Pichia pastoris, P.
  • methanolica P. stipitis, lipolytica Yarrowia lipolytica, Candida, Schwanniomyces occidentalis, Hansenula polymorpha, Saccharomyces carlella, Saccharomyces cerevisiae, Saccharomyces douglasa, Kluyveromyces nodii Yeast, ovoid yeast or combinations thereof.
  • the fungus is a filamentous fungus
  • the filamentous fungus is selected from the following group: Acremonium, Aspergillus, Aureobasidium, Tobacco Bjerkandera), Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium , Fusarium, Humicola, Pyrrhosporium, Magnaporthe, Mucor, Myceliophthora, Neomyceliophthora (Neocallimastix), Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleuromyces (Pleurotus), Schizophyllum, Talaromyces, Talaromyces, Thermoascus, Thielavia, Tolypocladium , Trametes, Trichoderma, Fusarium, Humicola, Neurospora, Scytalidium
  • the fungus is selected from the following group: Aspergillus terreus, Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, Aspergillus nidulans Aspergillus nidulans, Aspergillus fumigatus, Aspergillus aculeatus, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus oryzae, Rhizopus rhizopus (Rhizopus arrhizus), Rhizobus oryzae, Trichoderma reesei, Trichoderma reesei QM9414, Trichoderma reesei RUT-C30, Trichoderma reesei QM6a, Trichoderma atroviride ), T.harzianum, T.virens, T.a
  • the plant cell is a dicotyledonous plant cell or a monocotyledonous plant. cell.
  • the dicotyledonous plant cells are selected from the group consisting of soybean cells, sunflower cells, tomato cells, Brassica crop cells, cotton cells, sugar beet cells, tobacco cells, potato cells, petunia, and Arabidopsis thaliana. or combination thereof.
  • the monocot cell is selected from the group consisting of barley cells, maize cells, corn cells, oat cells, rice cells, sorghum cells, sugarcane cells, wheat cells, or combinations thereof.
  • the animal cells are insect cells or mammalian cells.
  • the insect cells are selected from the following group: Autographa californica (alfalfa looper), Spodoptera frugiperda (grassland) Spodoptera exigua (beet armyworm), Trichoplusia ni (cabbagelooper), Lymantria dispar (gypsy moth), Bombyx mori (silkworm), Anticarsia gemmatalis (velvetbeancaterpillar), Heliothis virescens (tobacco night moth) Tobacco budworm), Heliothis subflexa (Subflexus straw moth), Mamestra brassicae (cabbage moth), Helicoverpa armigera (cotton bollworm), corn earworm (Helicoverpa zea) (corn earworm), Agrotis ipsilon (black cutworm), Anagrapha falcifera (celery looper), Large Galleria mellonella (honeycomb moth), Rachiplusia ou (graylooper
  • the insect cells are selected from the following group: Sf9 cells from Spodoptera frugiperda, Sf21 cells from Spodoptera frugiperda, Trichopodia exigua High-Five cells from Trichoplusiani (same as Hi5, same as High-Five BTI-TN-5B1-4), Tn-368 cells from Trichoplusiani, from Spondoptera exigua Se301 cells, S2 cells from Drosophila melanogaster, Bm5 cells from Bombyx mori, Ld652Y from gypsy moth, LdEIta from gypsy moth, or combinations thereof.
  • the mammalian cells are selected from the following group: SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC CRL1651); human embryonic kidney cell line (HEK293 or suspension cultured 293 cells Subcloning, Graham et al., J. Gen Virol. 36: 59 (1977)), such as Expi293; baby hamster kidney cells (BHK, ATCC CCL 10); baby hamster kidney cells (BHK); Chinese hamster ovary cells/-DHFR ( CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse testicular Sertoli cells (TM4, Mather, Biol. Reprod.
  • COS-7 SV40 transformed monkey kidney cell CV1 line
  • baby hamster kidney cells BHK, ATCC CCL
  • monkey kidney cells CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver Cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., AnnalsN .Y.Acad.Sci.383:44-68(1982)); MRC 5 cells; FS4 cells; CHO cells; NSO cells; myeloma cell lines such as YB 2/0, YO, NS0, P3X63 and Sp2/0, etc. ; Lymphocytes (e.g., Y0, NSO, Sp20 cells); or combinations thereof.
  • CV1 ATCC CCL70 African green monkey kidney
  • the mammalian cells are selected from human cells, and the human human cells are selected from the following group: HeLa, Huh7, HEK293, HepG2, KATO-III, IMR32, MT-2, pancreatic ⁇ -cells, keratinocytes, bone marrow fibroblasts, CHP212, primary neural cells, W12, SK-N-MC, Saos-2, WI38, primary hepatocytes, FLC4, 143TK-, DLD-1, embryonic lung cells Fibrocytes, primary foreskin fibroblasts, Saos-2 osteosarcoma, MRC5, MG63 cells or combinations thereof.
  • the human human cells are selected from the following group: HeLa, Huh7, HEK293, HepG2, KATO-III, IMR32, MT-2, pancreatic ⁇ -cells, keratinocytes, bone marrow fibroblasts, CHP212, primary neural cells, W12, SK-N-MC, Sa
  • the fifth aspect of the present invention provides a method for preparing gene editing protein variants, the method includes the steps:
  • a sixth aspect of the present invention provides an enzyme preparation, which includes the gene editing protein variant described in the first aspect of the present invention.
  • the enzyme preparation includes injection and/or lyophilized preparation.
  • a seventh aspect of the present invention provides a gene editing system, including:
  • the gene editing protein variant according to the first aspect of the present invention or its encoding gene or its expression vector;
  • guide RNA or its expression vector, and/or its oligonucleotide or nucleic acid fragment or plasmid used for target site break repair are optionally provided.
  • the expression vector includes plasmid and viral vector.
  • the guide RNA includes crRNA, tracrRNA, and sgRNA.
  • the guide RNA includes unmodified and modified gRNA.
  • the modified guide RNA includes chemical modification of bases.
  • the chemical modification includes methylation modification, methoxy modification, fluorination modification or thiomodification.
  • the gene editing includes CRISPR-based gene editing.
  • An eighth aspect of the present invention provides a gene editing reagent, which contains the gene editing protein variant described in the first aspect of the present invention.
  • the reagents further include the following reagents:
  • Guide RNA or a vector used to produce said guide RNA, and/or its oligonucleotide or nucleic acid fragment or plasmid for target site break repair.
  • a ninth aspect of the present invention provides a composition comprising:
  • the composition includes a pharmaceutical composition.
  • the dosage form of the composition is selected from the following group: freeze-dried preparations, liquid preparations, or combinations thereof.
  • the dosage form of the composition is a liquid preparation.
  • the dosage form of the composition is an injection dosage form.
  • the composition is a cell preparation.
  • the expression vector of the gene editing protein variant and the expression vector of the guide RNA are the same vector or different vectors.
  • the system described in the third aspect of the present invention accounts for 1-99wt% of the total weight of the composition, preferably 10-90wt%, more preferably 30-70wt %.
  • a tenth aspect of the present invention provides a product combination, including:
  • the gene editing protein variant according to the first aspect of the present invention, the system according to the seventh aspect of the present invention, or the gene editing reagent according to the eighth aspect of the present invention is provided.
  • the product combination also includes: guide RNA, or a vector used to produce the guide RNA, and/or its oligonucleotide or nucleic acid fragment or plasmid for target site break repair. .
  • the product combination further includes a pharmaceutically acceptable carrier.
  • the eleventh aspect of the present invention provides a kit, comprising: the gene editing protein variant described in the first aspect of the present invention or the enzyme preparation described in the sixth aspect of the present invention or the gene editing described in the seventh aspect of the present invention.
  • the kit further includes a label or instructions.
  • a twelfth aspect of the present invention provides a medicine box, including:
  • the medicine in the first container contains the gene editing protein variant described in the first aspect of the present invention or the enzyme preparation described in the sixth aspect of the present invention or the seventh aspect of the present invention.
  • the dosage form of the drug is selected from the following group: freeze-dried preparations, liquid preparations, or combinations thereof.
  • the dosage form of the drug is an oral dosage form or an injection dosage form.
  • the kit also contains instructions.
  • a thirteenth aspect of the present invention provides a medicine box, including:
  • first container and the second container are different containers.
  • the drug in the first container is a single preparation containing the gene editing protein variant described in the first aspect of the present invention, or its encoding gene or its expression vector.
  • the drug in the second container contains guide RNA or its expression vector. Single preparation.
  • the dosage form of the drug is selected from the following group: freeze-dried preparations, liquid preparations, or combinations thereof.
  • the dosage form of the drug is an oral dosage form or an injection dosage form.
  • the kit also contains instructions.
  • the fourteenth aspect of the present invention provides a gene editing protein variant according to the first aspect of the present invention or the enzyme preparation according to the sixth aspect of the present invention or the gene editing system according to the seventh aspect of the present invention or the second aspect of the present invention.
  • the reagent or kit is used to reduce the trans-cleavage activity of gene editing.
  • the reagent or kit is used to reduce the trans-cleaving activity of gene editing while retaining the cis-cleaving activity.
  • reducing the trans-cleaving activity of gene editing means reducing the trans-cleaving activity of gene editing by ⁇ 80%, more preferably, by ⁇ 90% or 100%.
  • the fifteenth aspect of the present invention provides a method for reducing the off-target rate of gene editing, including the steps:
  • the cell is a prokaryotic cell or a eukaryotic cell.
  • the cells are mammalian cells.
  • the mammalian cells are non-human mammals, such as primates, cattle, sheep, porcines, dogs, rodents, Leporidae, such as monkeys, cows, sheep, pigs, dogs, Rabbit, rat or mouse cells.
  • non-human mammals such as primates, cattle, sheep, porcines, dogs, rodents, Leporidae, such as monkeys, cows, sheep, pigs, dogs, Rabbit, rat or mouse cells.
  • the cells are non-mammalian eukaryotic cells such as cells of poultry birds (eg chicken), vertebrate fish (eg salmon) or crustaceans (eg oysters, clams, lobsters, shrimps).
  • poultry birds eg chicken
  • vertebrate fish eg salmon
  • crustaceans eg oysters, clams, lobsters, shrimps.
  • the cells are plant cells.
  • the plant cell is a cell of a monocotyledonous plant or a dicotyledonous plant or a cell of a cultivated plant or a food plant such as cassava, corn, sorghum, soybean, wheat, oat or rice. cells.
  • the plant cell is an algae, a tree or a production plant, a fruit or a vegetable (for example, a tree such as a citrus tree, such as an orange tree, a grapefruit tree or a lemon tree; a peach or nectarine tree; an apple or pear tree; nut tree such as almond or walnut tree or pistachio tree; nightshade; brassica; lettuce; spinach; capsicum; cotton, tobacco, asparagus, carrot, cabbage , broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.).
  • a fruit or a vegetable for example, a tree such as a citrus tree, such as an orange tree, a grapefruit tree or a lemon tree; a peach or nectarine tree; an apple or pear tree; nut tree such as almond or walnut tree or pistachio tree; nightshade; brassica; lettuce; spinach; capsicum; cotton, tobacco, asparagus, carrot, cabbage , broccoli, cauliflower, tomato
  • the gene editing is performed in an in vitro reaction system.
  • the method is non-diagnostic and non-therapeutic.
  • the cells are cells in vitro.
  • Figure 1 is a gel diagram of gene editing protein purification, showing the molecular weight of three gene editing proteins, all of which are 150KDa.
  • Lanes 1 and 2 are wild-type FnCas12a, and the loading amounts are 3 ⁇ g and 5 ⁇ g respectively;
  • lanes 3 and 4 are mutant protein FnCas12a K1069R , and the loading amounts are 2 ⁇ g and 3 ⁇ g respectively;
  • lanes 5 and 6 are mutant proteins FnCas12a F1081R , and the upper The sample amounts were 2 ⁇ g and 3 ⁇ g respectively.
  • Figure 2 is an electrophoresis diagram of the cis-cleaving reaction product between the gene editing protein and the target dsDNA. It shows that the three proteins have cis-cleaving activity, and the cis-cleaving activity of the mutant proteins FnCas12a K1069R and FnCas12a F1081R is the same as that of wild-type gene editing. There was no significant difference in the cis-cleaving activity of the proteins.
  • M is the 1Kb DNA Marker
  • S is the target dsDNA fragment with a size of 829bp
  • P is the cis-cleavage product of the target dsDNA with sizes of 529bp and 300bp respectively.
  • Figure 3 shows the fluorescence signal changes in the trans-cleavage reaction between gene editing proteins and non-target ssDNA.
  • a real-time fluorescence quantitative PCR instrument was used to detect the fluorescence signal of the reaction system.
  • control is the negative control trans-cleavage reaction system, that is, no target dsDNA is added to the trans-cleavage system.
  • WT is the wild-type FnCas12a protein, and the fluorescence signal of its trans-cleavage reaction system increases with the extension of reaction time, indicating that the wild-type FnCas12a has trans-cleaving activity.
  • the fluorescence signal of the mutant protein FnCas12a K1069R and FnCas12a F1081R trans-cleavage reaction system has been at the background level and remained unchanged as the reaction time prolongs, indicating that the mutant proteins FnCas12a K1069R and FnCas12a F1081R have no significant trans-cleavage activity.
  • Figure 4 (4a-4e) is an alignment analysis of the amino acid sequences of 10 types of Cas12a proteins. It can be seen from this figure that the amino acid sequences of these 10 Cas12a proteins have high homology.
  • FIG. 5 is the evolutionary tree of CRISPR V-type Cas protein (i.e. Cas12 protein). As shown in the figure, Cas12 proteins all contain RuvC functional domains. (Yan Winston X et al. Functionally diverse type V CRISPR-Cas systems. [J]. Science (New York, N.Y.), 2018, 363(6422).)
  • Figure 6 is a schematic diagram of the protein domain of FnCas12a, indicating the starting and ending positions of the amino acid residues of each functional domain (Stefano, Stella, Pablo, et al. Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity.[J]. Cell, 2018, 175: 1856-1871).
  • Figure 7 is a schematic diagram of the protein domains of Cas12a, Cas12b, and Cas12e (Tong Baisong et al. The Versatile Type V CRISPR Effectors and Their Application Prospects[J]. Frontiers in Cell and Developmental Biology, 2021, 8: 622103-622103.)
  • the inventor After extensive and in-depth research, the inventor originally tried to mutate the effector protein of the V-type family in order to increase its interaction with the trans-cleavage active substrate DNA. After extensive screening, on the contrary, he unexpectedly obtained a gene editing protein variant. .
  • the gene editing protein variant of the present invention can have cis-cleaving activity and reduced trans-cleaving activity, or even no trans-cleaving activity, and the gene editing protein variant of the present invention and the protein containing the present invention
  • Gene editing systems based on gene editing protein variants can significantly reduce the off-target rate of gene editing. On this basis, the inventor completed the present invention.
  • the term “about” may refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as in this article As used, the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term “contains” or “includes” can be open, semi-closed and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
  • Sequence identity is measured along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein ) is determined by comparing two aligned sequences and determining the number of positions where identical residues occur. Typically, this is expressed as a percentage.
  • the measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.
  • cis-cleaving activity refers to the specific cleavage activity of Cas protein on target nucleic acid molecules.
  • trans-cleavage activity refers to the non-specific cleavage activity of Cas protein on non-target nucleic acid molecules (mainly non-target single-stranded nucleic acid molecules).
  • wild-type gene editing protein refers to a naturally occurring gene editing protein that has not been artificially modified, and its nucleotides can be obtained through genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR) ), etc., the amino acid sequence of which can be derived from the nucleotide sequence.
  • the sources of the wild-type gene editing proteins include Lachnospiraceae bacterium ND2006 (LbCas12a), Thiomicrospira sp.
  • BoCas12a BoCas12a
  • Oribacterium sp.NK2B42 OsCas12a
  • Acidaminococcus sp.BV3L6 AsCas12a
  • Helcococcus sp kunzii HkCas12a
  • Lachnospiraceae bacterium NC2008 Lb5Cas12a.
  • the protein includes Cas12, Cas14, and further includes Cas12a, Cas12b, and Cas12e; further, the Cas12a is selected from the following group: FnCas12a, LbCas12a, ErCas12a, Evcas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, CeCas 12a, PrCas12a , CsbCas12a, BhCas12a, SsCas12a, Lb3Cas12a, BpCas12a, PdCas12a, BfCas12a, PcCas12a, cMtCas12a, PeCas12a, LiCas12a, Lb2Cas12a, PmCas12a
  • the wild-type gene editing protein is FnCas12a, and the sequence is shown in SEQ ID NO. 1.
  • gene editing protein variant As used herein, the terms "gene editing protein variant”, “variant of the invention”, “gene editing mutein of the invention” and “mutein” are used interchangeably and refer to non-binary proteins with cis-cleaving activity.
  • a naturally occurring mutated gene editing protein, and the mutant protein is mutated at one or more core amino acid positions related to cleavage activity of the wild-type gene editing protein selected from the following group:
  • core amino acid refers to a gene-edited protein based on wild-type and at least 80% homology with the wild-type gene-edited protein, such as 84%, 85%, 90%, 92%, 95%, 98% Or in 99% of the sequences, the corresponding position is the specific amino acid described in this article.
  • the core amino acid is:
  • mutant protein obtained by mutating the above-mentioned core amino acids has cis-cleaving activity and reduced trans-cleaving activity, or even no trans-cleaving activity.
  • the core amino acid of the present invention is mutated as follows:
  • the phenylalanine (F) corresponding to position 1081 of FnCas12a was mutated to arginine (R);
  • Lysine (K) corresponding to position 1069 of FnCas12a was mutated to arginine (R).
  • the amino acid numbering in the mutant protein of the present invention is based on the wild-type gene editing protein.
  • the amino acid number of the mutant protein may be misaligned relative to the amino acid numbering of the wild-type gene-edited protein, such as a misalignment of 1-100 positions toward the N-terminus or C-terminus of the amino acid.
  • mutant proteins with reduced trans-cleavage activity are not within the scope of the mutant proteins of the present invention.
  • the mutant protein of the present invention is a synthetic protein or a recombinant protein, that is, it can be a product of chemical synthesis or produced from a prokaryotic or eukaryotic host (eg, bacteria, yeast, plant) using recombinant technology.
  • a prokaryotic or eukaryotic host eg, bacteria, yeast, plant
  • the muteins of the invention may be glycosylated or may be non-glycosylated.
  • the muteins of the invention may or may not include an initial methionine residue.
  • the present invention also includes fragments, derivatives and analogs of said muteins.
  • fragment refers to proteins that retain substantially the same biological function or activity of the mutant protein.
  • the mutein fragments, derivatives or analogs of the present invention may be (i) a mutein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acids
  • the residues may or may not be encoded by the genetic code, or (ii) a mutein with substituent groups in one or more amino acid residues, or (iii) a mature mutein combined with another compound such as an elongated mutein A mutein formed by fusion of a half-life compound, such as polyethylene glycol, or (iv) an additional amino acid sequence fused to the mutein sequence (such as a leader sequence or secretion sequence or used to purify the mutein) sequence or protein sequence, or a fusion protein formed with an antigenic IgG fragment).
  • conservatively substituted amino acids are preferably produced by amino acid substitution
  • the active mutant protein of the present invention has cis-cleaving activity and reduced trans-cleaving activity, or even no trans-cleaving activity.
  • the mutant protein is as shown in any one of SEQ ID NO.: 2-3.
  • the mutant protein of the present invention usually has higher homology (identity) than the sequence shown in any one of SEQ ID NO.: 2-3.
  • the mutant protein has higher homology (identity) with SEQ ID NO.
  • the homology of any sequence shown in NO.: 2-3 is at least 80%, preferably at least 85%-90%, more preferably at least 95%, optimally at least 98% or 99% .
  • mutant protein of the present invention can also be modified.
  • Modified (usually without changing primary structure) forms include chemically derivatized forms of the mutant protein such as acetylation or carboxylation in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of the mutein during its synthesis and processing or during further processing steps. This modification can be accomplished by exposing the mutant protein to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include those with phosphorylated amino acid residues such as phosphotyrosine acid, phosphoserine, phosphothreonine) sequence. Also included are mutant proteins that have been modified to increase their resistance to proteolysis or to optimize solubility.
  • polynucleotide encoding a mutein may include polynucleotides encoding the mutein of the present invention, or may also include polynucleotides that additionally include coding and/or non-coding sequences.
  • sequence of the polynucleotide encoding the mutant protein of the present invention is as shown in any one of SEQ ID NO.: 4-5.
  • FnCas12a K1069R nucleotide sequence (SEQ ID NO.4):
  • FnCas12a F1081R nucleotide sequence (SEQ ID NO.5):
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or mutant proteins having the same amino acid sequence as the present invention.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide, which may be the substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the mutant protein it encodes. Function.
  • the invention also relates to polynucleotides that hybridize to the sequences described above and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize under stringent conditions (or stringent conditions) to the polynucleotides of the invention.
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, more It is best when hybridization occurs only when the ratio is above 95%.
  • the muteins and polynucleotides of the invention are preferably provided in isolated form and, more preferably, are purified to homogeneity.
  • the full-length sequence of the polynucleotide of the present invention can usually be obtained through PCR amplification, recombination or artificial synthesis.
  • primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and commercially available cDNA libraries or cDNA prepared by conventional methods known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.
  • the DNA sequence encoding the protein of the present invention (or its fragments, or its derivatives) can be obtained entirely through chemical synthesis.
  • the DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the invention through chemical synthesis.
  • the method of amplifying DNA/RNA using PCR technology is preferably used to obtain the polynucleotide of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-rapid amplification of cDNA ends) can be preferably used.
  • the primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods.
  • the amplified DNA/RNA fragments can be separated and purified using conventional methods such as by gel electrophoresis.
  • the present invention also relates to vectors containing the polynucleotide of the present invention, as well as host cells genetically engineered using the vector of the present invention or the mutant protein coding sequence of the present invention, and methods for producing the polypeptides of the present invention through recombinant technology.
  • polynucleotide sequences of the present invention can be used to express or produce recombinant mutant proteins by conventional recombinant DNA techniques. Generally speaking there are the following steps:
  • the polynucleotide sequence encoding the mutant protein can be inserted into the recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and be stable in the host body.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translation control elements.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding the mutant proteins of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.
  • the DNA sequence can be effectively linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters include: E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture.
  • GFP Fluorescent protein
  • tetracycline or ampicillin resistance in E. coli tetracycline or ampicillin resistance in E. coli.
  • Vectors containing appropriate DNA sequences as described above and appropriate promoter or control sequences can be used to transform appropriate host cells to enable expression of proteins.
  • the host cell can be a prokaryotic cell (such as Escherichia coli), a lower eukaryotic cell, or a higher eukaryotic cell, such as a yeast cell, a plant cell or a mammalian cell (including human and non-human mammals).
  • a prokaryotic cell such as Escherichia coli
  • a lower eukaryotic cell such as a yeast cell
  • a plant cell such as a mammalian cell (including human and non-human mammals).
  • mammalian cell including human and non-human mammals.
  • Representative examples include: Escherichia coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, yeast cells, etc.
  • yeast cells such as Pichia pastoris, Kluyveromyces cerevisiae, or combinations thereof are selected; preferably, the yeast cells include: Kluyveromyces marxianus, more preferably Kluyveromyces marxianus Kluyveromyces lactis and/or Kluyveromyces lactis) is the host cell.
  • Enhancers are DNA cis-acting factors, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method.
  • the steps used are well known in the art.
  • Another method is to use MgCl2.
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in culture can be selected from various conventional media. Cultivate under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed within the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the present invention discovers a new gene editing protein variant for the first time.
  • the gene editing protein variant of the invention can have cis-cleaving activity and reduced trans-cleaving activity, or even no trans-cleaving activity. Cleaving activity, and the gene editing protein variant of the present invention and the gene editing system containing the gene editing protein variant of the present invention can significantly reduce the off-target rate of gene editing.
  • Design primers containing mutation sites use wild-type FnCas12a expression plasmid as template, use Phanta DNA polymerase to amplify linear fragments with target site mutations, and use Ezmax (obtained from Anhui Tolo Port Biotechnology Co., Ltd.
  • the constructed pET28TEV-FnCas12a plasmid was transformed into E.coli BL21 (DE3) competent cells, and cultured in solid LB medium containing 50 ⁇ g/mL kana (hereinafter referred to as Kan) resistance at 37°C for 12-14 hours. Pick 3 single clones and add them to 50 mL of Kan-resistant liquid LB medium. After culturing overnight on a shaker at 37°C, transfer 1% (v/v) to 1L of Kan-resistant liquid LB medium.
  • Kan solid LB medium containing 50 ⁇ g/mL kana
  • AMED16s-F/R (sequence AMED16s-F: 5′-gtgaactaagccagtagagc-3′, AMED16s-R: 5′-ctttcgctcctcagcgtcag-3′, synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.) was used as the amplification primer.
  • the genome of Amycobacterium mediterranea U32 (NCBI accession number: SAMN02603409) was used as a template for PCR amplification.
  • the PCR amplification system of the target dsDNA fragment is shown in Table 2.
  • the PCR reaction program is: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 57°C for 15 s, extension at 72°C for 30 s (1 min can amplify 2 kb), 32 cycles, and finally, extension at 75°C for 5 min. 1.5% (w/v) agarose gel electrophoresis was used to identify the size of the fragment.
  • the amplification product was a correct single DNA fragment.
  • the column recovery method was used to recover the target fragment. Promega's Wizard SV Gel and PCR clean-up system reagent was used for column recovery. box.
  • crRNA sequence 5′-AAUUUCUACUCUUGUAGAUGCCAGGGACGAAGCGCAAGUGACGGAAU-3′, synthesized by Nanjing Genscript Biotechnology Co., Ltd. and purified by HPLC.
  • the detection method is as follows: react at 37°C for 40 minutes, inactivate at 85°C for 5 minutes, and add a final concentration of 1 ⁇ DNA loading. All reaction products were loaded, electrophoresed on 2% (w/v) agarose gel, electrophoresed at 140V for 25 minutes, stained with EB for 30 minutes, and photographed with a gel imager. The cis-cleaving products were about 529 bp and 300 bp DNA fragments.
  • Control s experimental system does not add FnCas12a protein. The experimental results are shown in Figure 2.
  • HOLMES-P (FQ-reporter), purchased from Anhui Toluang Biotechnology Co., Ltd., is a short single-stranded DNA probe modified with a FAM fluorescent luminescent group on one end and a fluorescent quenching group on the other end (5'-TTTTTT- 3′).
  • FAM fluorescent luminescent group on one end
  • fluorescent quenching group on the other end
  • the DNA probe does not fluoresce; only when the single-stranded DNA fragment is cut and the quenching group is separated from the fluorescent group, the fluorescence of the DNA probe can be detected. Signal.
  • the present invention analyzes the structure of FnCas12a.
  • the amino acids of FnCas12a that interact with the DNA substrate include: K1069, F1081, F1010, V1285, N1288, etc. These amino acid sites may be related to trans-cleavage activity.
  • the present invention mutates these sites and measures the cis- and trans-cleaving activities of these proteins. Finally, two mutant proteins with cis-cleaving activity and no trans-cleaving activity are obtained.
  • the mutations of these two proteins are respectively The amino acid at position 1081 is mutated from phenylalanine to arginine (F1081R) and the amino acid at position 1069 is mutated from lysine to arginine (K1069R).
  • the corresponding protein names are FnCas12a F1081R and FnCas12a K1069R respectively.
  • WT wild-type protein
  • mutant proteins F1081R and K1069R
  • the cis-cleaving activity test results showed that there was no significant difference between the cis-activities of the two mutant proteins, FnCas12a F1081R and FnCas12a K1069R , and FnCas12a ( Figure 2).
  • the trans-cleavage activity detection results showed that the trans-cleavage activity of FnCas12a F1081R and FnCas12a K1069R was significantly reduced compared with the trans-cleavage activity of wild-type FnCas12a protein ( Figure 3).
  • the present invention has discovered two mutant proteins of FnCas12a. Their mutation sites are the mutation of amino acid 1081 from phenylalanine to arginine (F1081R) and the mutation of amino acid 1069 from lysine to arginine (F1081R). Arginine (K1069R), these two mutant proteins retain the cis-cleaving activity and lose (or significantly reduce) the trans-cleaving activity of the original wild-type gene editing. Since the Cas12a wild-type protein not only It can specifically cleave target DNA and also has non-specific trans-cleaving activity on single-stranded DNA, which will cause a certain degree of off-target effects during the gene editing process.
  • the trans-cleaving activity of Cas12a is removed (or reduced) by artificially modifying the wild-type gene editing protein while retaining its cis-cleaving activity, overcoming the problems caused by the trans-cleaving activity of the gene editing protein. off-target problem, thus giving the Cas12a mutant protein more advantages in gene editing.
  • type 2 clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are characterized by a single effector protein and can be further subdivided into types II, V, and VI. Effector proteins of the V-type family are diverse at the N-terminus but retain a unified RuvC-like endonuclease domain at the C-terminus.
  • the V-type system is further subdivided into many subtypes, including type V-A to type V-I, type V-K, type V-U, and CRISPR-Cas8 ⁇ (see Figure 5).
  • Cas12a (V-A type), Cas12b (V-B type) and Cas12e (V-E type) all belong to the V-type system.
  • the effector protein binds to gRNA to form a binary complex, they specifically recognize 5'-T-rich PAM and promote The target DNA unwinds, and at the same time, the non-target strand (NTS) of the target sequence is displaced, forming a so-called "R-loop" structure.
  • the RuvC domain sequentially cleaves the NTS and target strand (TS) distal to the PAM, forming a staggered cut with 5, 7, or 10 NT strand 5′ overhangs.
  • Cas12a, Cas12b, and Cas12e these three proteins have a bilobed structure composed of an ⁇ -helical recognition (REC) lobe and a nuclease (NUC) lobe (see Figure 7).
  • the two lobes are connected by a bridge-helix (BH) domain.
  • the REC lobe contains two REC domains (REC1 and REC2), which mainly help regulate and stabilize the hybridization of crRNA target and DNA after forming an "R loop".
  • site 1069 of FnCas12a is located in the RuvC domain.
  • Each Cas protein under the V-type system has a RuvC domain (see Figure 5) and corresponding sites. It can be expected that mutation of the amino acid residue corresponding to the 1069 position of FnCas12a in each Cas protein under the V-type system will produce similar results.
  • site 1081 of FnCas12a is located at the junction of the RuvC domain and the NUC domain.
  • Cas12a, Cas12b, and Cas12e all have RuvC domains and NUC domains (see Figure 7). It can be expected that mutation of the amino acid residue corresponding to the 1081 position of FnCas12a in Cas12a, Cas12b, and Cas12e, which have similar structures and functions and high homology, will achieve similar effects; especially those with more similar structures and functions. Highly sourced Similar effects will be obtained by mutating the amino acid residue corresponding to position 1081 of FnCas12a in Cas12a from other sources (see Figure 4, ae) (see Table II).

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Abstract

本发明提供一种可降低基因编辑脱靶率的基因编辑蛋白变体，所述变体为具有顺式切割活性的非天然蛋白，且所述变体相较于其野生型基因编辑蛋白的反式切割活性降低，并且所述变体在野生型基因编辑蛋白的选自下组一个或多个与切割活性相关的核心氨基酸位点发生突变：对应于FnCas12a第1081位的苯丙氨酸(F)位点；和/或对应于FnCas12a第1069位的赖氨酸(K)位点，该变体可具有顺式切割活性且反式切割活性降低，并且本发明的基因编辑蛋白变体以及含有本发明的基因编辑蛋白变体的基因编辑系统可显著降低基因编辑脱靶率。

Description

可降低基因编辑脱靶率的基因编辑蛋白变体 技术领域
本发明涉及生物技术领域，具体地，涉及可降低基因编辑脱靶率的基因编辑蛋白变体。
背景技术
基因编辑是指对DNA序列进行删除、插入或替换等操作，广泛应用于基因功能研究、疾病模型建立、疾病治疗以及转基因动植物工程等等。第一代基因编辑技术基于锌指核酸酶(Zinc Finger Nuclease，ZFN)，ZFN含有一个能够特异性识别序列的DNA锌指结合域，通过改造这个区域可以实现靶向不同的DNA序列。一个DNA锌指结合域一般由多个锌指结构组成，每个锌指结构识别3个碱基，因此ZNF的靶序列必须是3的倍数。由于ZNF的识别结构域存在上下文依赖效应，其设计和筛选的难度非常大，应用范围受到限制，并且该技术还存在成本高、劳动量大、耗时长、成功率低、易脱靶、细胞毒性大等缺陷。第二代基因编辑技术基于转录激活样效用因子核酸酶(TALEN，Transcription Activator-like effector Nuclease)，其识别靶位点DNA特异性的单位模块是间隔32个恒定氨基酸残基的二联氨基酸，不同的二联氨基酸能够与AGTC四个核苷酸碱基一一对应。根据靶标DNA的序列反推出对应的二联氨基酸序列，从而构成TALEN靶点识别模块。该模块进行组装需要大量的分子克隆和测序操作，从而限制了该技术的推广。
第三基因编辑技术基于CRISPR-Cas技术，该技术通过向导RNA(guide RNA)实现特异性识别靶标DNA序列，guide RNA的设计和合成工作量远小于TALEN和ZFN技术的DNA识别模块的构建过程。Guide RNA能够结合具有核酸酶活性的Cas蛋白，并引导其对靶标DNA进行切割。
目前基因编辑蛋白还是存在一定程度的脱靶率。当基因编辑蛋白(比如Cas12a)与向导RNA和靶标DNA形成三元复合体后，不仅对靶标DNA具有顺式切割活性，还对体系中存在的单链DNA具有非特异的反式切割活性。当DNA处于复制或转录状态时，双链DNA会解链成单链DNA，此时基因编辑蛋白(比如Cas12a)的反式切割活性可能会导致这些DNA被切割，因而导致脱靶产生，并且引起细胞毒问题。因此需要消除基因编辑蛋白(比如Cas12a)的 反式切割活性以解决脱靶引起的细胞毒问题。
因此，本领域迫切需要开发消除基因编辑蛋白(比如Cas12a)的反式切割活性以解决脱靶引起的细胞毒问题的方法。
发明内容
本发明的目的是提供一种降低乃至消除基因编辑蛋白(比如Cas12a)的反式切割活性以解决脱靶引起的细胞毒问题的方法。
本发明第一方面提供了一种基因编辑蛋白变体，所述变体为具有顺式切割活性的非天然蛋白，且所述变体相较于其野生型基因编辑蛋白的反式切割活性降低，并且所述变体在野生型基因编辑蛋白的选自下组一个或多个核心氨基酸位点发生突变：
对应于FnCas12a第1081位的苯丙氨酸(F)位点；和/或
对应于FnCas12a第1069位的赖氨酸(K)位点。
在另一优选例中，所述变体相较于其野生型基因编辑蛋白的反式切割活性降低指与野生型的基因编辑蛋白相比，所述变体的反式切割活性降低≥50％，较佳地≥80％，更佳地，≥90％或100％。
在另一优选例中，FnCas12a第1081位的苯丙氨酸(F)突变为选自下组的一种或多种氨基酸：精氨酸(R)、酪氨酸(Y)、色氨酸(W)、谷氨酰胺(Q)、天冬酰胺(N)、赖氨酸(K)、谷氨酸(E)、天冬氨酸(D)或其组合。
在另一优选例中，FnCas12a第1069位的赖氨酸(K)突变为选自下组的一种或多种氨基酸：精氨酸(R)、酪氨酸(Y)、谷氨酰胺(Q)、天冬酰胺(N)、赖氨酸(K)、谷氨酸(E)、天冬氨酸(D)或其组合。
在另一优选例中，FnCas12a第1081位的苯丙氨酸(F)突变为精氨酸(R)。
在另一优选例中，FnCas12a第1069位的赖氨酸(K)突变为精氨酸(R)。
在另一优选例中，所述的突变选自下组：F1081R、K1069R、或其组合。
在另一优选例中，所述基因编辑蛋白为V型CRISPR/Cas蛋白。
在另一优选例中，所述基因编辑蛋白选自下组：Cas 12、Cas14、或其组合。
在另一优选例中，所述基因编辑蛋白选自下组：Cas12a、Cas12b、Cas12e或其组合。
另一优选例中，所述Cas12a选自下组：FnCas12a、LbCas12a、ErCas12a、Evcas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a、Lb4Cas12a、CeCas12a、PrCas12a、CsbCas12a、BhCas12a、SsCas12a、Lb3Cas12a、BpCas12a、PdCas12a、BfCas12a、PcCas12a、cMtCas12a、PeCas12a、LiCas12a、Lb2Cas12a、PmCas12a、MbCas12a、EeCas12a、CsbCas12a、ArCas12a、BsCas12a、AbCas12a、AsCas12a、或其组合。
在另一优选例中，所述Cas12a的来源选自下组：纤毛菌属、李斯特菌属、棒状杆菌属、萨特氏菌属、军团菌属、密螺旋体属、产线菌属、真细菌属、链球菌属、乳酸菌属、支原体属、拟杆菌属、Flaviivola、黄杆菌属、固氮螺菌属、Sphaerochaeta、葡糖醋杆菌属、奈瑟氏菌属、罗氏菌属、Parvibaculum、葡萄球菌属、Nitratifractor、支原体属、弯曲杆菌属、毛螺菌属、或其组合。
在另一优选例中，所述Cas12a的来源选自下组：图拉弗朗西斯菌(Francisella tularensis)(FnCas12a)、氨基酸球菌属BV3L6(Acidaminococcus sp.BV3L6)(AsCas12a)、毛螺菌科细菌ND2006(Lachnospiraceae bacterium ND2006)(LbCas12a)、毛螺菌科细菌NC2008(Lachnospiraceae bacterium NC2008)(Lb5Cas12a)、孔氏创伤球菌(Helcococcus sp kunzii)(HkCas12a)、Oribacterium sp.NK2B42(OsCas12a)、Thiomicrospira sp.XS5(TsCas12a)、拟杆菌KA00251(Bacteroidales bacterium KA00251)(BbCas12a)、口腔拟杆菌(Bacteroidetes oral taxon 274)(BoCas12a)、毛螺菌科细菌MC2017(Lachnospiraceae bacterium MC2017)(Lb4Cas12a)、规则粪球菌(Coprococcus eutactus)(CeCas12a)、栖瘤胃普氏菌(Prevotella ruminicola strain BPI-34)(PrCas12a)、Candidatus Saccharibacteria bacterium(CsbCas12a)、亨氏丁酸弧菌(Butyrivibrio hungatei strain MB2003)(BhCas12a)、史密斯氏菌SC_K08D17(Smithella sp.SC_K08D17)(SsCas12a)、毛螺菌科细菌MC2017(Lachnospiraceae bacterium MC2017)(Lb3Cas12a)、瘤胃溶纤维丁酸弧菌(Bytyrivibrio proteoclasticus)(BpCas12a)、普氏杆菌(Prevotella disens)(PdCas12a)、溶纤维丁酸弧菌MD2001(Butyrivibrio fibrisolvens MD2001)(BfCas12a)、狗口腔卟啉单胞菌(Porphyromonas crevioricanis)PcCas12a、Candidatus Methanoplasma termitum(CMtCas12a)、异域菌门细菌(Peregrinibacteria bacterium)(PeCas12a)、Leptospira inadaiserovar Lyme(LiCas12a)、毛螺菌科细菌MA2020(Lachnospiraceae bacterium MA2020)(Lb2Cas12a)、猕猴卟啉单胞菌(Porphyromonas  macaca)(PmCas12a)、牛莫拉氏菌(Moraxella bovoculi 237)(MbCas12a)、挑剔真杆菌(Eubacterium eligens)(EeCas12a)、Candidatus Saccharibacteria bacterium(CsbCas12a)、直肠真杆菌(Eubacte riumrectale)(ErCas12a)、直肠酵母菌(Agathobacter rectalisstrain)(ArCas12a)、丁酸弧菌NC3005(Butyrivibrio sp.NC3005)(BsCas12a)、布氏弓形杆菌(Arcobacter butzleri)(AbCas12a)或其组合。
在另一优选例中，所述Cas12b的来源选自下组：卡克氏脂环酸芽孢杆菌(Alicyclobacillus kakegawensis)、芽孢杆菌属V3-13种、外村尚芽孢杆菌(Bacillus hisashii)、黏胶球形菌纲细菌(Lentisphaeria bacterium)、沉积物莱西氏菌(Laceyella sediminis)或其组合。
在另一优选例中，所述Cas12b选自下组：AacCas12b、AaCas12b、BthCas12b、AapCas12b、AkCas12b、AmCas12b、Bs3Cas12b、LsCas12b或其组合。
在另一优选例中，所述基因编辑蛋白的来源选自下组：纤毛菌属、李斯特菌属、棒状杆菌属、萨特氏菌属、军团菌属、密螺旋体属、产线菌属、真细菌属、链球菌属、乳酸菌属、支原体属、拟杆菌属、Flaviivola、黄杆菌属、固氮螺菌属、Sphaerochaeta、葡糖醋杆菌属、奈瑟氏菌属、罗氏菌属、Parvibaculum、葡萄球菌属、Nitratifractor、支原体属、弯曲杆菌属、毛螺菌属、或其组合。
在另一优选例中，所述基因编辑蛋白的来源选自下组：毛螺菌科细菌ND2006(Lachnospiraceae bacterium ND2006)(LbCas12a)、Thiomicrospira sp.XS5(TsCas12a)、土拉弗菌(Francisella tularensis)(FnCas12a)、Bacteroidetes oral taxon 274(BoCas12a)、Oribacterium sp.NK2B42(OsCas12a)、氨基酸球菌属BV3L6(Acidaminococcus sp.BV3L6)(AsCas12a)、孔氏创伤球菌(Helcococcus sp kunzii)(HkCas12a)、毛螺菌科细菌NC2008(Lachnospiraceae bacterium NC2008)(Lb5Cas12a)、或其组合。
在另一优选例中，所述FnCas12a第1081和1069位的位点位于FnCas12a的第1081和1069位。
在另一优选例中，所述FnCas12a第1081和1069位的位点位于BbCas12a的第1019和1007位。
在另一优选例中，所述FnCas12a第1081和1069位的位点位于AsCas12a的第1069和1057位。
在另一优选例中，所述FnCas12a第1081和1069位的位点位于BoCas12a的第1033和1021位。
在另一优选例中，所述FnCas12a第1081和1069位的位点位于HkCas12a的第1090和1078位。
在另一优选例中，所述FnCas12a第1081和1069位的位点位于Lb4Cas12a的第1004和992位。
在另一优选例中，所述FnCas12a第1081和1069位的位点位于Lb5Cas12a的第980和968位。
在另一优选例中，所述FnCas12a第1081和1069位的位点位于LbCas12a的第1018和1006位。
在另一优选例中，所述FnCas12a第1081和1069位的位点位于OsCas12a的第1001和989位。
在另一优选例中，所述FnCas12a第1081和1069位的位点位于TsCas12a的第1070和1058位。
在另一优选例中，所述基因编辑蛋白为FnCas12a。
在另一优选例中，所述基因编辑蛋白的序列如SEQ ID NO.1所示。
在另一优选例中，所述变体的氨基酸序列如SEQ ID NO.2-3中的任一所示。
在另一优选例中，所述的变体为具有SEQ ID NO.：2-3中任一所示氨基酸序列的多肽、其活性片段、或其保守性变异多肽。
在另一优选例中，所述的变体除所述突变(如1081位、和/或1069位)外，其余的氨基酸序列与野生型的基因编辑蛋白的序列相同或基本相同。
在另一优选例中，所述的基本相同是至多有50个(较佳地为1-20个，更佳地为1-10个、更佳地1-5个)氨基酸不相同，其中，所述的不相同包括氨基酸的取代、缺失或添加，且所述的变体具有顺式切割活性且反式切割活性降低。
在另一优选例中，所述变体与所述野生型的基因编辑蛋白的同源性至少为80％，较佳地至少为85％或90％，更佳地至少为95％，最佳地至少为98％或99％。
在另一优选例中，所述的变体选自下组：
(a)具有SEQ ID NO.：2-3中任一所示氨基酸序列的多肽；
(b)将SEQ ID NO.：2-3中任一所示氨基酸序列经过一个或多个(如2个、3个、4个或5个)氨基酸残基的取代、缺失或添加而形成的，且具有顺式切割活性且反式切割活性降低的由(a)衍生的多肽。
在另一优选例中，所述的衍生的多肽与SEQ ID NO.：2-3中任一所示序列的同 源性至少为60％，较佳地至少为70％，更佳地至少为80％，最佳地至少为90％，如95％、97％、99％。
在另一优选例中，所述变体为所述野生型的基因编辑蛋白经突变形成的。
本发明第二方面提供了一种多核苷酸，所述的多核苷酸编码本发明第一方面所述的变体。
在另一优选例中，所述多核苷酸选自下组：
(a)编码如SEQ ID NO.2-3中任一所示多肽的多核苷酸；
(b)序列如SEQ ID NO.：4-5中任一所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.：4-5中任一所示序列的同源性≥80％(较佳地≥90％，更佳地≥95％，最佳地≥98％)，且编码SEQ ID NO.：2-3中任一所示多肽的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述的多核苷酸在所述变体的ORF的侧翼还额外含有选自下组的辅助元件：信号肽、分泌肽、标签序列(如6His)、或其组合。
在另一优选例中，所述的多核苷酸选自下组：基因组序列、cDNA序列、RNA序列、或其组合。
在另一优选例中，该多核苷酸还包含与所述变体的ORF序列操作性连接的启动子。
在另一优选例中，所述的启动子选自下组：组成型启动子、组织特异性启动子、诱导型启动子、或者强启动子。
本发明第三方面提供了一种载体，所述的载体含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体包含一个或多个启动子，所述启动子可操作地与所述核酸序列、增强子、转录终止信号、多腺苷酸化序列、复制起点、选择性标记、核酸限制性位点、和/或同源重组位点连接。
在另一优选例中，所述载体包括质粒载体、噬菌体载体、cosmid克隆载体、噬菌粒载体、人工染色体载体、附加型载体、病毒载体或其组合。
在另一优选例中，所述人工染色体载体选自下组：细菌人工染色体(BAC)、酵母人工染色体(YAC)、P1人工染色体(PAC)或其组合；
在另一优选例中，所述病毒载体选自下组：逆转录病毒载体、腺病毒载体、 腺相关病毒载体、疱疹病毒载体、痘病毒载体、杆状病毒载体、乳头多瘤空泡病毒、乳头瘤病毒载体、HBP爱巴病毒(HBP Epstein Barrvirus)载体、牛痘病毒载体、塞姆利基森林病毒(Semliki Forest virus；SFV)或其组合；
优选地，所述载体选自下组：pcDNA、pTT、pTT3、pEFBOS、pBV、pJV、pBJ、pGEX、VSV、pBR322、pCMV-HA、pEN、YAC、BAC、λ噬菌体、M13噬菌体、噬菌粒、pCAS9、pCEN6、pYES1L、p3HPRT1、pFN2A、pBC、pTZ、pGEM、pGEMK、pEX、pSAR、pCEP、黏性质粒、pBluescript、pKJK、pFloxin、pCP、pHR、pUC、pMAL、pALTER、pBAD、pCal、pL、pET、pGEMEX、pCI、pCMV、pEGFP、pEGFT、pSV2、pFUSE、pVITRO、pVIVO、pMONO、pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS420、pLexA、pACT2.2、pRS403、pRS404、pRS405、pRS406、pRS413、pRS414、pRS415、pRS416或其组合。
在另一优选例中，所述载体包括克隆载体、转化载体、表达载体、穿梭载体、整合载体、多功能载体。
本发明第四方面提供了一种宿主细胞，所述宿主细胞含有本发明第三方面所述的载体，或其基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞为原核受体细胞。
在另一优选例中，进一步，所述原核受体细胞选自下组：大肠杆菌、乳酸菌、枯草杆菌、蓝细菌、链霉菌(Streptomyces)、假单胞菌、丙酸杆菌属(Propionibacterium)、梳状菌属(Pectinatus sp.)、拟杆菌属(Bacteroides sp.)、枯草芽孢杆菌(Bacillus subtilis)、链霉菌属(Streptomyces)、鱼腥藻属(Anabaena)、节杆菌属(Arthrobacter)、农杆菌属(Agrobacterium)、醋杆菌属(Acetobacter)、醋酸杆菌属(Acetobacterium)、芽孢杆菌属(Bacillus)、短芽孢杆菌属(Brevibacillus)、双歧杆菌属(Bifidobacterium)、短状杆菌属(Brachybacterium)、短杆菌属(Brevibacterium)、肉杆菌属(Carnobacterium)、致病杆菌属(Xenorhabdus)、发光杆菌属(Photorhabdus)、棒状杆菌属(Corynebacterium)、肠杆菌属(Enterobacter)、巴氏杆菌属(Pasteurella)、乳杆菌属(Lactobacillus)、产碱杆菌属(Alcaligenes)、黄杆菌属(Flavobacterium spp.)、梭菌属(Clostridium)、巴斯德氏芽菌属(Pasteuria)、埃希氏菌属(Escherichia)、葡糖酸醋酸杆菌属(Gluconacetobacter)、 葡糖杆菌属(Gluconobacter)、哈夫尼菌属(Hafnia)、嗜盐单胞菌属(Halomonas)、克雷伯氏菌属(Klebsiella)、考克氏菌属(Kocuria)、明串珠菌属(Leucononstoc)、巨大球菌属(Macrococcus)、甲基单胞菌属(Methylomonas)、甲基细菌属(Methylobacter)、甲基细胞菌属(Methylocella)、甲基球菌属(Methylococcus)、微杆菌属(Microbacterium)、微球菌属(Micrococcus)、微囊菌属(Microcystis)、穆尔氏菌属(Moorella)、酒球菌属(Oenococcus)、片球菌属(Pediococcus)、原绿球藻属(Prochlorococcus)、丙酸杆菌属(Propionibacterium)、变形杆菌属(Proteus)、假交替单胞菌属(Pseudoalteromonas)、假单胞菌属(Pseudomonas)、嗜冷菌属(Psychrobacter)、红细菌属(Rhodobacter)、红球菌属(Rhodococcus)、红假单胞菌属(Rhodopseudomonas)、欧文氏菌属(Erwinia)、志贺氏菌属(shigella)、沙雷氏菌属(Serratia)、沙门氏菌属(Salmonella)、葡萄球菌属(Staphylococcus)、链球菌属(Streptococcus)、链霉菌属(Streptomyces)、根瘤菌属(Rhizobium)、聚球藻菌属(Synechococcus)、集胞藻属(Synechocystis)、四联球菌属(Tetragenococcus)、魏斯氏菌属(Weissella)、黄单胞菌属(Xanthomonas)、发酵单胞菌属(Zymomonas)、红假单胞菌属(Rhodopseudomonas)、以及鼠伤寒沙门氏菌(Salmonellatyphimuium)、嗜甲基菌属(Methylophilius)、固氮菌属(Azotobacter)、剑菌属(Ensifer)、鞘脂单胞菌属、伯克氏菌属、Candidatus Glomeribacter、戴氏菌属、草螺菌属、慢生根瘤菌属、贪噬菌属、鞘氨醇杆菌属(Sphingobacterium)、单胞发酵菌属、沙雷菌属、气单孢菌属、弧菌属、脱硫弧菌属、螺旋菌属、醋菌属或其组合。
在另一优选例中，进一步，原核受体细胞选自下组：大肠杆菌(Escherichia Coli.)、类球红细菌(Rodhobacter sphaeroides)、游海假交替单胞菌(Pseudoalteromonas haloplanktis)、希瓦氏菌属(Shewanella sp.)菌株Ac10、荧光假单胞菌(Pseudomonas fluorescens)、恶臭假单胞菌(Pseudomonas putida)、铜绿假单胞菌(Pseudomonas aeruginosa)、产碱假单胞菌(P.alcaligenes)、铜绿假单胞菌PAO1-LAC、恶臭假单胞菌KT2440、伸长盐单胞菌(Halomonas elongata)、柠檬假交替单胞菌(Pseudoalteromonas citrea)、需盐色盐杆菌(Chromohalobacter salex’igens)、变铅青链霉菌(Streptomyces lividans)、灰色链霉菌(Streptomyces griseus)、天蓝链霉菌(Streptomyces coelicolor)、除虫链霉菌(S.avermitilis)、灰色链霉菌(S.griseus)、疮痂链霉菌(S.scabies)、浅青紫链霉菌(S.lividans)TK24、浅 青紫链霉菌1326、耐内酰胺诺卡氏菌(Nocardia lactamdurans)、耻垢分枝杆菌(Mycobacterium smegmatis)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、产氨棒状杆菌(Corynebacterium ammoniagenes)、乳糖发酵短杆菌(Brevibacteriumlactofermentum)、烟草节杆菌(Arthrobacter nicotianae)、纹膜醋酸杆菌(Acetobacter aceti)、阿氏节杆菌(Arthrobacter arilaitensis)、蜡样芽孢杆菌(Bacillus cereus)、凝结芽孢杆菌(Bacillus coagulans)、球形芽孢杆菌(Bacillus sphaericus)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)、枯草芽孢杆菌(Bacillus subtilis)、短芽孢杆菌(Bacillus brevis)、巨大芽孢杆菌(Bacillus megaterium)、地衣芽孢杆菌(Bacillus licheniformis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、乳酸乳球菌(Lactococcus lactis)、植物乳杆菌(Lactobacillus plantarum)、干酪乳杆菌(Lactobacillus casei)、罗伊氏乳杆菌(Lactobacillus reuteri)、加氏乳杆菌(Lactobacillus gasseri)、酸面乳杆菌(Lactobacillus acidifarinae)、山梨乳杆菌(Lactobacillusyamanashiensis)、詹氏乳杆菌(Lactobacillusjensenii)、沙克乳杆菌(Lactobacillus sakei)、氧化葡糖杆菌(Gluconobacter oxydans)、青春双歧杆菌(Bifidobacterium adolescentis)、奶酪发酵小短杆菌(Brachybacteriumtyrofermentans)、扩展短杆菌(Brevibacterium linens)、广布肉毒杆菌(Carnobacteriumdivergens)、微黄棒状杆菌(Corynebacterium flavescens)、欧洲葡糖酸醋酸杆菌(Gluconacetobacter europaeus)、约氏葡糖酸醋酸杆菌(Gluconacetobacter johannae)、氧化葡糖杆菌(Gluconobacter oxydans)、产酸克氏杆菌(Klebsiella oxytoca)、产琥珀酸放线杆菌(Actinobacillus succinogenes)、产琥珀酸曼氏杆菌(Mannhei succiniciproducers)MBEL 55E、嗜淀粉拟杆菌(Bacteroidesamylophilus)、小叶微杆菌(Microbacterium foliorum)、产丙酸丙酸杆菌(Propionibacterium acidipropionici)、普通变形杆菌(Proteus vulgaris)、速生嗜冷杆菌(Psychrobacter celer)、比菲德氏/短/长双歧杆菌(Bifidobacterium bifidum/breve/longum)、蜂房哈夫尼菌(Hafnia alvei)、产琥珀酸厌氧螺菌(Anaerobiospirillum succiniciproducens)、生黄瘤胃球菌(Ruminococcus flavefaciens)、栖瘤胃普雷沃氏菌(Prevotella ruminicola)、溶淀粉琥珀酸单胞菌(Succcinimonas amylolytica)、溶糊精琥珀酸弧菌(Succinivibrio dextrinisolvens)、产琥珀酸沃林氏菌(Wolinellasuccinogenes)、产琥珀酸噬纤维菌(Cytophaga succinicans)、菜豆根瘤 菌(Rhizobium etli)、运动单胞发酵菌(Zymomonas mobilis)、丙酮丁醇梭菌(Clostridium acetobutylicum)、杨氏/醋酸/丙酮丁醇/拜氏/丁酸梭菌(Clostridium ljungdahlii/aceticum/acetobutylicum/beijerinckii/butyricum)、屎肠球菌(Enterococcus faecium)、里拉微球菌(Micrococcuslylae)、酒类酒球菌(Oenococcus oeni)、溶酪大球菌(Macrococcus caseolyticus)、乳酸片球菌(Pediococcus acidilactici)、香料葡萄球菌(Staphylococcus condimenti)、嗜热链球菌(Streptococcus thermophilus)、嗜盐四联球菌(Tetragenococcus halophilus)、嗜根考克氏菌(Kocuriarhizophila)、柠檬明串珠菌(Leuconostoc citreum)、食窦魏斯氏菌(Weissella cibaria)、高丽魏斯氏菌(Weissella koreensis)、嗜热纤维/热乙酸穆尔氏菌(Moorellathemocellum/thermoacetica)、苏云金芽孢杆菌(Bacillus thuringiensis)、或其组合。
在另一优选例中，更进一步，所述大肠杆菌选自下组：BL21、BL21(DE3)、W3110、MG1655、RB791、RV308、HMS 174、HMS174(DE3)、NM533、XL1-Blue、C600、DH1、HB101、JM109、Top10、DH5α、DH10β、TG1、BW23473、BW23474、MW003、MW005细胞或其组合；所述巨大芽孢杆菌(Bacillus megaterium)选自下组：QMB1551、PV361、DSM319或其组合。
在另一优选例中，所述的宿主细胞为真核细胞。
在另一优选例中，进一步，所述真核受体细胞选自下组：酵母、真菌、植物细胞、动物细胞或其组合。
在另一优选例中，又进一步，所述酵母选自以下种：红酵母属(Rhodotorula spp.)、短梗霉属物(Aureobasidium spp.)、酵母属(Saccharomyces spp.)、掷孢酵母属物种(Sporobolomyces spp.)、及其组合。
在另一优选例中，又进一步，所述酵母选自下组：酿酒酵母(Saccharomyces cerevisiae)、粟酒裂殖酵母(Schizosaccharomyces pombe)、乳酸克鲁维酵母(Kluyveromyces lactis)、脆壁克鲁维酵母(ATCC 12，424)、保加利亚克鲁维酵母(ATCC 16，045)、魏氏克鲁维酵母(ATCC 24，178)、克鲁雄酵母(ATCC 56，500)、瓦尔提鲁维酵母(K.waltii)(ATCC 56500)、果蝇克鲁维酵母(ATCC 36，906)、耐热克鲁维酵母、马克斯克鲁维酵母(Kluyveromyces marxianus)、巴斯德毕赤酵母(Pichia pastoris)、甲醇毕赤酵母(P.methanolica)、树干毕赤酵母(P.Stipitis)、解脂 耶罗维亚酵母(Yarrowia lipolytica)、假丝酵母、许旺酵母(Schwanniomyces occidentalis)、多形汉逊酵母(Hansenulapolymorpha)、卡尔酵母、糖化酶母、道格拉氏酵母、克鲁弗酵母、诺地酵母、卵形酵母或其组合。
在另一优选例中，又进一步，所述真菌是丝状真菌，所述丝状真菌选自下组：枝顶孢霉属、曲霉属、短梗霉属(Aureobasidium)、烟管霉属(Bjerkandera)、拟蜡菌属(Ceriporiopsis)、金孢子菌属(Chrysosporium)、鬼伞菌属(Coprinus)、革盖菌属(Coriolus)、隐球菌属(Cryptococcus)、线黑粉菌属(Filibasidium)、镰孢属(Fusarium)、腐质霉属(Humicola)、梨孢菌属、大角间座壳属(Magnaporthe)、毛霉属(Mucor)、毁丝霉属(Myceliophthora)、新美鞭菌属(Neocallimastix)、链孢菌属(Neurospora)、拟青霉属(Paecilomyces)、青霉属、平革菌属(Phanerochaete)、射脉菌属(Phlebia)、瘤胃壶菌属(Piromyces)、侧耳属(Pleurotus)、裂褶菌属(Schizophyllum)、踝节菌属(Talaromyces)、篮状菌属、嗜热子囊菌属(Thermoascus)、梭孢壳菌属(Thielavia)、弯颈霉属(Tolypocladium)、栓菌属(Trametes)、木霉属、镰刀霉属、腐殖菌属、链孢霉属、柱霉属(Scytalidium)、肉座菌属(Hypocrea)、金孢霉属、黑粉酵母属(Filibasidium)、赤霉菌属(Gibberella)、梨孢菌属(Magnaporthe)、毛霉属(Mucor)、毁丝霉属、漆斑菌属(Myrothecium)、新考玛脂霉属(Neocallimastix)或其组合。
在另一优选例中，又进一步，所述真菌选自下组：土曲霉(Aspergillus terreus)、米曲霉(A.oryzae)、黑曲霉(Aspergillus niger)、泡盛曲霉(A.awamori)、构巢曲霉(Aspergillus nidulans)、烟曲霉(Aspergillusfumigatus)、棘孢曲霉(Aspergillusaculeatus)、棒状曲霉(Aspergillusclavatus)、黄曲霉(Aspergillusflavus)、臭曲霉(Aspergillusfoetidus)、日本曲霉(Aspergillusjaponicus)、米曲霉、少根根霉菌(Rhizopus arrhizus)、米根霉菌(Rhizobus oryzae)、里氏木霉(Trichoderma reesei)、里氏木霉QM9414、里氏木霉RUT-C30、里氏木霉QM6a、深绿木霉(T.atroviride)、哈茨木霉(T.harzianum)、粘绿木霉(T.virens)、棘孢木霉(T.asperellum)、长枝木霉(T.longibrachiatum)、康氏木霉(Trichoderma koningii)、长梗木霉(Trichodermalongibrachiatum)、深绿木霉(Trichodermaatroviride)、绿木霉(Trichoderma virens)、绿色木霉 (Trichodermaviride)、宛氏拟青霉(Paecilomyces varioti)、葡萄酒青霉(Penicillium viniferum)、产紫青霉(P.purpurogenum)、绳状青霉(P.funiculosum)、埃默森青霉(篮状菌)(Penicillium(Talaromyces)emersonii)、沙门柏干酪青霉(P.camemberti)、娄地青霉(P.roqueforti)、黄孢原毛平革菌(Phanerochaete chrysosporium)、棉阿舒囊霉(Ashbya gossypii)、雪白丝衣霉(Byssochlamys nivea)、黑烟管菌(Bjerkandera adusta)、干拟蜡菌(Ceriporiopsis aneirina)、卡内基拟蜡菌(Ceriporiopsis caregiea)、浅黄拟蜡菌(Ceriporiopsis gilvescens)、潘诺希塔拟蜡菌(Ceriporiopsis pannocinta)、环带拟蜡菌(Ceriporiopsis rivulosa)、微红拟蜡菌(Ceriporiopsis subrufa)、虫拟蜡菌(Ceriporiopsis subvermispora)、卢克诺文思金孢子菌(Chrysosporium lucknowense)、狭边金孢子菌(Chrysosporium inops)、嗜角质金孢子菌(Chrysosporium keratinophilum)、粪状金孢子菌(Chrysosporium merdarium)、租金孢子菌(Chrysosporium pannicola)、昆士兰金孢子菌(Chrysosporium queenslandicum)、热带金孢子菌(Chrysosporium tropicum)、褐薄金孢子菌(Chrysosporium zonatum)、灰盖鬼伞(Coprinus cinereus)、毛革盖菌(Coriolushirsutus)、杆孢状镰孢(Fusarium bactridioides)、谷类镰孢(Fusarium cerealis)、库威镰孢(Fusarium crookwellense)、大刀镰孢(Fusarium culmorum)、禾谷镰孢(Fusarium graminearum)、禾赤镰孢(Fusarium graminum)、异孢镰孢(Fusarium heterosporum)、合欢木镰孢(Fusarium negundi)、尖孢镰孢(Fusarium oxysporum)、多枝镰孢(Fusarium reticulatum)、粉红镰孢(Fusarium roseum)、接骨木镰孢(Fusarium sambucinum)、肤色镰孢(Fusarium sarcochroum)、拟分枝孢镰孢(Fusarium sporotrichioides)、硫色镰孢(Fusarium sulphureum)、圆镰孢(Fusarium torulosum)、拟丝孢镰孢(Fusariumtrichothecioides)、镶片镰孢、特异腐质霉、柔毛腐质霉、米黑毛霉、嗜热毁丝霉、粗糙链孢菌(Neurospora crassa)、产紫青霉(Penicillium purpurogenum)、黄孢平革菌(Phanerochaete chrysosporium)、射脉菌(Phlebia radiata)、刺芹侧耳(Pleurotuseryngii)、土生梭孢壳霉(Thielavia terrestris)、长域毛栓菌(Trametes villosa)、变色栓菌(Trametes versicolor)、或其组合。
在另一优选例中，又进一步，所述植物细胞是双子叶植物细胞或单子叶植物 细胞。更进一步优选地，所述双子叶植物细胞选自下组：大豆细胞、向日葵细胞、番茄细胞、芸苔属作物细胞、棉花细胞、甜菜细胞、烟草细胞、马铃薯细胞、矮牵牛、拟南芥或其组合。所述单子叶植物细胞选自下组：大麦细胞、玉蜀黍细胞、玉米细胞、燕麦细胞、稻细胞、高粱细胞、甘蔗细胞、小麦细胞、或其组合。
在另一优选例中，又进一步，所述动物细胞是昆虫细胞或哺乳动物细胞。
在另一优选例中，更进一步，所述昆虫细胞选自下组：苜蓿银纹夜蛾(Autographa californica)(苜蓿环纹夜蛾(alfalfa looper))，草地贪夜蛾(Spodoptera frugiperda)(草地夜蛾(fall army worm))，甜菜夜蛾(Spodoptera exigua)(甜菜夜蛾(beet armyworm))，粉纹夜蛾(Trichoplusia ni)(粉纹夜蛾(cabbagelooper))，舞毒蛾(Lymantria dispar)(舞毒蛾(gypsy moth))，家蚕(Bombyx mori)(桑蚕(silkworm))，黎豆夜蛾(Anticarsia gemmatalis)(黎豆毛虫(velvetbeancaterpillar))，烟芽夜蛾(Heliothis virescens)(烟草夜蛾(tobacco budworm))，Heliothis subflexa(Subflexus straw moth)，甘蓝夜蛾(Mamestra brassicae)(甘蓝夜蛾(cabbage moth))，棉铃虫(Helicoverpa armigera)(棉铃虫(cotton bollworm))，玉米穗虫(Helicoverpa zea)(玉米穗虫(corn earworm))，球菜夜蛾(Agrotis ipsilon)(黑地老虎(black cutworm))，芹菜夜蛾(Anagrapha falcifera)(芹菜夜蛾(celery looper))，大蜡螟(Galleria mellonella)(蜂窝蛾(honeycomb moth))，Rachiplusia ou(graylooper)，小菜蛾(Plutella xylostella)(小菜蛾(diamondback moth))，黑腹果蝇(Drosophila melanogaster)(果蝇)，埃及伊蚊(Aedes aegypti)(蚊子)，或其组合。
在另一优选例中，更进一步，所述昆虫细胞选自下组：来自草地贪夜蛾(Spodoptera frugiperda)的Sf9细胞，来自草地贪夜蛾(Spodoptera frugiperda)的Sf21细胞，来自粉纹夜蛾(Trichoplusiani)的High-Five细胞(与Hi5相同，与High-Five BTI-TN-5B1-4相同)，来自粉纹夜蛾(Trichoplusiani)的Tn-368细胞，来自甜菜夜蛾(Spondoptera exigua)的Se301细胞，来自黑腹果蝇(Drosophila melanogaster)的S2细胞，来自家蚕的Bm5细胞，来自舞毒蛾的Ld652Y，来自舞毒蛾的LdEIta，或其组合。
在另一优选例中，更进一步，所述哺乳动物细胞选自下组：SV40转化的猴肾细胞CV1系(COS-7，ATCC CRL1651)；人胚胎肾细胞系(HEK293或悬浮培养的293细胞亚克隆，Graham等，J.Gen Virol.36：59(1977))，如Expi293；幼仓鼠肾细胞(BHK，ATCC CCL 10)；幼仓鼠肾细胞(BHK)；中国仓鼠卵巢细胞/-DHFR(CHO，Urlaub等，Proc.Natl.Acad.Sci.USA 77：4216(1980))；小鼠睾丸支持细胞(TM4，Mather，Biol.Reprod.23：243-251(1980))；猴肾细胞(CV1 ATCC CCL70)；非洲绿猴肾细胞(VERO-76，ATCC CRL-1587)；人宫颈癌细胞(HELA，ATCC CCL 2)；犬肾细胞(MDCK，ATCC CCL 34)；布法罗大鼠肝细胞(BRL 3A，ATCC CRL 1442)；人肺细胞(W138，ATCC CCL 75)；人肝细胞(HepG2，HB 8065)；小鼠乳腺瘤(MMT 060562，ATCC CCL51)；TRI细胞(Mather等，AnnalsN.Y.Acad.Sci.383：44-68(1982))；MRC 5细胞；FS4细胞；CHO细胞；NSO细胞；骨髓瘤细胞系如YB 2/0、YO、NS0、P3X63和Sp2/0等；淋巴细胞(例如，Y0、NS0、Sp20细胞)；或其组合。
在另一优选例中，更进一步，所述哺乳动物细胞选自人类细胞，所述人人类细胞选自下组：HeLa、Huh7、HEK293、HepG2、KATO-III、IMR32、MT-2、胰腺β-细胞、角化细胞、骨髓成纤维细胞、CHP212、原代神经细胞、W12、SK-N-MC、Saos-2、WI38、原代肝细胞、FLC4、143TK-、DLD-1、胚胎肺成纤维细胞、原代包皮成纤维细胞、Saos-2骨肉瘤、MRC5、MG63细胞或其组合。
本发明第五方面提供了一种基因编辑蛋白变体的制备方法，所述的方法包括步骤：
(a)在适合表达的条件下，培养本发明第四方面所述的宿主细胞，从而表达所述的基因编辑蛋白变体；和
(b)分离所述的基因编辑蛋白变体。
本发明第六方面提供了一种酶制剂，所述酶制剂包括本发明第一方面所述的基因编辑蛋白变体。
在另一优选例中，所述的酶制剂包括注射剂、和/或冻干制剂。
本发明第七方面提供了一种基因编辑系统，包括：
本发明第一方面所述的基因编辑蛋白变体、或其编码基因或其表达载体；和
任选的，向导RNA或其表达载体，和/或其用于靶标位点断裂修复的寡核苷酸或核酸片段或质粒。
在另一优选例中，所述表达载体包括质粒、病毒载体。
在另一优选例中，所述向导RNA包括crRNA、tracrRNA、sgRNA。
在另一优选例中，所述向导RNA包括未修饰和经修饰的gRNA。
在另一优选例中，所述经修饰的向导RNA包括碱基的化学修饰。
在另一优选例中，所述化学修饰包括甲基化修饰、甲氧基修饰、氟化修饰或硫代修饰。
在另一优选例中，所述的基因编辑包括基于CRISPR的基因编辑。
本发明第八方面提供了一种基因编辑试剂，所述基因编辑试剂包含本发明第一方面所述的基因编辑蛋白变体。
在另一优选例中，所述的试剂还包括如下试剂：
向导RNA、或用于产生所述向导RNA的载体，和/或其用于靶标位点断裂修复的寡核苷酸或核酸片段或质粒。
本发明第九方面提供了一种组合物，包括：
本发明第一方面所述的基因编辑蛋白变体或本发明第七方面所述的系统或本发明第八方面所述的基因编辑试剂；和
药学上可接受的载体。
在另一优选例中，所述组合物包括药物组合物。
在另一优选例中，所述组合物的剂型选自下组：冻干制剂、液体制剂、或其组合。
在另一优选例中，所述组合物的剂型为液体制剂。
在另一优选例中，所述组合物的剂型为注射剂型。
在另一优选例中，所述组合物为细胞制剂。
在另一优选例中，所述基因编辑蛋白变体的表达载体和向导RNA的表达载体为同一载体或不同载体。
在另一优选例中，所述的组合物中，本发明第三方面所述的系统占所述组合物总重量的1-99wt％，较佳地10-90wt％，更佳地30-70wt％。
本发明第十方面提供了一种产品组合，包括：
本发明第一方面所述的基因编辑蛋白变体或本发明第七方面所述的系统或本发明第八方面所述的基因编辑试剂。
在另一优选例中，所述的产品组合还包括：向导RNA、或用于产生所述向导RNA的载体，和/或其用于靶标位点断裂修复的寡核苷酸或核酸片段或质粒。
在另一优选例中，所述产品组合还包括药学上可接受的载体。
本发明第十一方面提供了一种试剂盒，包括：本发明第一方面所述的基因编辑蛋白变体或本发明第六方面所述的酶制剂或本发明第七方面所述的基因编辑系统或本发明第八方面所述的基因编辑试剂或本发明第九方面所述的组合物或本发明第十方面所述的产品组合。
在另一优选例中，所述试剂盒还包括标签或说明书。
本发明第十二方面提供了一种药盒，包括：
第一容器，以及位于所述第一容器中的本发明第一方面所述的基因编辑蛋白变体或本发明第六方面所述的酶制剂或本发明第七方面所述的基因编辑系统或本发明第八方面所述的基因编辑试剂或本发明第九方面所述的组合物或本发明第十方面所述的产品组合，或含有本发明第一方面所述的基因编辑蛋白变体或本发明第六方面所述的酶制剂或本发明第七方面所述的基因编辑系统或本发明或本发明第八方面所述的基因编辑试剂或本发明第九方面所述的组合物或本发明第十方面所述的产品组合的药物。
在另一优选例中，所述的第一容器的药物是含本发明第一方面所述的基因编辑蛋白变体或本发明第六方面所述的酶制剂或本发明第七方面所述的基因编辑系统或本发明第八方面所述的基因编辑试剂或本发明第九方面所述的组合物或本发明第十方面所述的产品组合的单方制剂。
在另一优选例中，所述药物的剂型选自下组：冻干制剂、液体制剂、或其组合。
在另一优选例中，所述药物的剂型为口服剂型或注射剂型。
在另一优选例中，所述的药盒还含有说明书。
本发明第十三方面提供了一种药盒，包括：
(a1)第一容器，以及位于所述第一容器中的本发明第一方面所述的基因编辑蛋白变体、或其编码基因或其表达载体，或含有本发明第一方面所述的基因编辑蛋白变体、或其编码基因或其表达载体的药物；
(b1)任选的第二容器，以及位于所述第二容器中的向导RNA或其表达载体，或含有向导RNA或其表达载体的药物。
在另一优选例中，所述的第一容器和第二容器为不同的容器。
在另一优选例中，所述的第一容器的药物是含本发明第一方面所述的基因编辑蛋白变体、或其编码基因或其表达载体的单方制剂。
在另一优选例中，所述的第二容器的药物是含向导RNA或其表达载体的 单方制剂。
在另一优选例中，所述药物的剂型选自下组：冻干制剂、液体制剂、或其组合。
在另一优选例中，所述药物的剂型为口服剂型或注射剂型。
在另一优选例中，所述的药盒还含有说明书。
本发明第十四方面提供了一种本发明第一方面所述的基因编辑蛋白变体或本发明第六方面所述的酶制剂或本发明第七方面所述的基因编辑系统或本发明第八方面所述的基因编辑试剂或本发明第九方面所述的组合物或本发明第十方面所述的产品组合或本发明第十三方面或第十四方面所述的药盒的用途，用于制备用于降低基因编辑脱靶率的试剂或试剂盒。
在另一优选例中，所述试剂或试剂盒用于降低基因编辑的反式切割活性。
在另一优选例中，所述试剂或试剂盒用于降低基因编辑的反式切割活性同时保留顺式切割活性。
在另一优选例中，所述降低基因编辑的反式切割活性指将基因编辑的反式切割活性降低≥80％，更佳地，≥90％或100％。
本发明第十五方面提供了一种降低基因编辑脱靶率的方法，包括步骤：
在本发明第一方面所述的基因编辑蛋白变体或本发明第六方面所述的酶制剂或本发明第七方面所述的基因编辑系统或本发明第八方面所述的基因编辑试剂或本发明第九方面所述的组合物或本发明第十方面所述的产品组合或本发明第十三方面或第十四方面所述的药盒的存在下，对细胞进行基因编辑，从而降低基因编辑脱靶率。
在另一优选例中，所述细胞是原核细胞或真核细胞。
在另一优选例中，所述细胞是哺乳动物细胞。
在另一优选例中，所述哺乳动物细胞是非人类哺乳动物，例如灵长类动物、牛、羊、猪类、犬、啮齿动物、兔科，例如猴、母牛、绵羊、猪、狗、兔、大鼠或小鼠的细胞。
在另一优选例中，所述细胞是非哺乳动物真核细胞例如家禽鸟类(例如鸡)、脊椎动物鱼(例如鲑鱼)或甲壳类动物(例如牡蛎、蛤、龙虾、虾)的细胞。
在另一优选例中，所述细胞是植物细胞。
在另一优选例中，所述植物细胞是单子叶植物或双子叶植物具有的细胞或栽培植物或粮食植物例如木薯、玉米、高粱、大豆、小麦、燕麦或稻具有的细 胞。
在另一优选例中，所述植物细胞是藻类、树或生产植物、果实或蔬菜(例如，树类例如柑橘树，例如桔子树、葡萄柚树或柠檬树；桃树或油桃树；苹果树或梨树；坚果树例如杏树或核桃树或阿月浑子树；茄属植物；芸苔属植物；莴苣属植物；菠菜属植物；辣椒属植物；棉花、烟草、芦笋、胡萝卜、甘蓝、西兰花、花椰菜、番茄、茄子、胡椒、莴苣、菠菜、草莓、蓝莓、覆盆子、黑莓、葡萄、咖啡、可可等)具有的细胞。。
在另一优选例中，所述的基因编辑在一体外反应体系中进行。
在另一优选例中，所述方法为非诊断性和非治疗性的。
在另一优选例中，所述细胞为体外的细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为基因编辑蛋白纯化胶图，显示了三种基因编辑蛋白的分子量大小，均为150KDa。泳道1和2为野生型FnCas12a，上样量分别为3μg和5μg；泳道3和4为突变型蛋白FnCas12aK1069R，上样量分别为2μg和3μg；泳道5和6为突变型蛋白FnCas12aF1081R，上样量分别为2μg和3μg。
图2为基因编辑蛋白与靶标dsDNA的顺式切割反应产物的电泳图，显示了三种蛋白都有顺式切割活性，且突变体蛋白FnCas12aK1069R和FnCas12aF1081R的顺式切割活力与野生型基因编辑蛋白的顺式切割活性相比并没有显著差异。图2中M为1Kb DNA Marker；S为靶标dsDNA片段，大小为829bp；P为靶标dsDNA的顺式切割产物，大小分别为529bp和300bp。
图3为基因编辑蛋白与非靶标ssDNA的反式切割反应的荧光信号变化图。如图3所示，用实时荧光定量PCR仪器检测反应体系的荧光信号。其中，control为阴性对照反式切割反应体系，即反式切割体系中不添加target dsDNA。随着时间的增加，control体系中没有检测到荧光信号。WT为野生型FnCas12a蛋白，其反式切割反应体系的荧光信号随着反应时间的延长而增强，说明野生型 FnCas12a具有反式切割活性。突变体蛋白FnCas12aK1069R、FnCas12aF1081R反式切割反应体系的荧光信号随着反应时间的延长，一直处于本底水平，保持不变，说明突变体蛋白FnCas12aK1069R和FnCas12aF1081R没有显著的反式切割活性。
图4(4a-4e)是10种类型的Cas12a蛋白氨基酸序列比对分析图。从该图可以得知，这10种Cas12a蛋白氨基酸序列具有较高的同源性。
图5是CRISPR V型Cas蛋白(即Cas12蛋白)的进化树。据图所示，Cas12蛋白都含有RuvC功能结构域。(Yan Winston X等人.Functionally diverse type V CRISPR-Cas systems.[J].Science(New York，N.Y.)，2018，363(6422).)。
图6是FnCas12a的蛋白质结构域示意图，标明了各功能结构域的氨基酸残基起止位置(Stefano，Stella，Pablo，等人.Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity.[J].Cell，2018，175：1856-1871)。
图7是Cas12a、Cas12b、Cas12e的蛋白质结构域示意图(Tong Baisong等人.The Versatile Type V CRISPR Effectors and Their Application Prospects[J].Frontiers in Cell and Developmental Biology，2021，8：622103-622103.)
具体实施方式
本发明人经过广泛而深入的研究，原本尝试突变V型家族的效应蛋白以期增加其与反式切割活性底物DNA的相互作用，经过大量筛选，却相反地意外获得一种基因编辑蛋白变体。相比野生型基因编辑蛋白，本发明的基因编辑蛋白变体可具有顺式切割活性且反式切割活性降低，甚至没有反式切割活性，并且本发明的基因编辑蛋白变体以及含有本发明的基因编辑蛋白变体的基因编辑系统可显著降低基因编辑脱靶率。在此基础上，本发明人完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文 所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由...构成”、或“由...构成”。
序列同一性(或同源性)通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
顺式切割活性
在本发明中，顺式切割活性是指Cas蛋白对靶标核酸分子的特异切割活性。
反式切割活性
在本发明中，反式切割活性是指Cas蛋白对非靶标核酸分子(主要是非靶标单链核酸分子)的非特异切割活性。
当DNA处于复制或转录状态时，双链DNA会解链成单链DNA，此时基因编辑蛋白(比如Cas12a)的反式切割活性可能会导致这些单链状态的DNA被切断，从而引起脱靶切割，因此，降低基因编辑蛋白的反式切割活性等同于降低基因编辑蛋白的基因编辑脱靶率。
野生型的基因编辑蛋白
如本文所用，“野生型的基因编辑蛋白”是指天然存在的、未经过人工改造的基因编辑蛋白，其核苷酸可以通过基因工程技术来获得，如基因组测序、聚合酶链式反应(PCR)等，其氨基酸序列可由核苷酸序列推导而得到。所述野生型基因编辑蛋白的来源包括毛螺菌科细菌ND2006(Lachnospiraceae bacterium ND2006)(LbCas12a)、Thiomicrospira sp.XS5(TsCas12a)、土拉弗菌(Francisella tularensis)(FnCas12a)、Bacteroidetes oral taxon 274(BoCas12a)、Oribacterium sp.NK2B42(OsCas12a)、氨基酸球菌属BV3L6(Acidaminococcus sp.BV3L6)(AsCas12a)、孔氏创伤球菌(Helcococcus sp kunzii)(HkCas12a)、毛螺菌科细菌NC2008(Lachnospiraceae bacterium NC2008)(Lb5Cas12a)。野生型的基因编辑 蛋白包括Cas12、Cas14，进一步包括Cas12a、Cas12b、Cas12e；又进一步，所述Cas12a选自以下组：FnCas12a、LbCas12a、ErCas12a、Evcas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a、Lb4Cas12a、CeCas12a、PrCas12a、CsbCas12a、BhCas12a、SsCas12a、Lb3Cas12a、BpCas12a、PdCas12a、BfCas12a、PcCas12a、cMtCas12a、PeCas12a、LiCas12a、Lb2Cas12a、PmCas12a、MbCas12a、EeCas12a、CsbCas12a、ArCas12a、BsCas12a、AbCas12a、AsCas12a、或其组合。
在本发明的一个优选例中，所述野生型的基因编辑蛋白为FnCas12a，序列如SEQ ID NO.1所示。
野生型FnCas12a氨基酸序列(SEQ ID NO.1)：

基因编辑蛋白变体及其编码核酸
如本文所用，术语“基因编辑蛋白变体”、“本发明的变体”、“本发明的基因编辑突变蛋白”、“突变蛋白”均可互换使用，均指具有顺式切割活性的非天然存在的突变的基因编辑蛋白，并且所述突变蛋白在野生型的基因编辑蛋白的选自下组的一个或多个与切割活性相关的核心氨基酸位点发生突变：
对应于FnCas12a第1081位的苯丙氨酸(F)位点；和/或
对应于FnCas12a第1069位的赖氨酸(K)位点，且所述突变蛋白相较于其野生型基因编辑蛋白的反式切割活性降低，甚至没有反式切割活性。
术语“核心氨基酸”指的是基于野生型的基因编辑蛋白，且与野生型的基因编辑蛋白同源性达至少80％，如84％、85％、90％、92％、95％、98％或99％的序列中，相应位点是本文所述的特定氨基酸，如基于野生型的基因编辑蛋白，核心氨基酸为：
对应于FnCas12a第1081位的苯丙氨酸(F)；和/或
对应于FnCas12a第1069位的赖氨酸(K)。
且对上述核心氨基酸进行突变所得到的突变蛋白具有顺式切割活性且反式切割活性降低，甚至没有反式切割活性。
优选地，在本发明中，对本发明的所述核心氨基酸进行如下突变：
对应于FnCas12a第1081位的苯丙氨酸(F)突变为精氨酸(R)；
对应于FnCas12a第1069位的赖氨酸(K)突变为精氨酸(R)。
应理解，本发明突变蛋白中的氨基酸编号基于野生型的基因编辑蛋白作出，当某一具体突变蛋白与野生型的基因编辑蛋白的序列的同源性达到80％或以上时，突变蛋白的氨基酸编号可能会有相对于野生型的基因编辑蛋白的氨基酸编号的错位，如向氨基酸的N末端或C末端错位1-100位，而采用本领域常规的序列比对技术，本领域技术人员通常可以理解这样的错位是在合理范围内的，且不应当由于氨基酸编号的错位而使同源性达80％(如90％、95％、98％)的、具有相同或相似的具有顺式切割活性且反式切割活性降低的突变蛋白不在本发明突变蛋白的范围内。
本发明突变蛋白是合成蛋白或重组蛋白，即可以是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、植物)中产生。根据重组生产方案所用的宿主，本发明的突变蛋白可以是糖基化的，或可以是非糖基化的。本发明的突变蛋白还可包括或不包括起始的甲硫氨酸残基。
本发明还包括所述突变蛋白的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持所述突变蛋白相同的生物学功能或活性的蛋白。
本发明的突变蛋白片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的突变蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的突变蛋白，或(iii)成熟突变蛋白与另一个化合物(比如延长突变蛋白半衰期的化合物，例如聚乙二醇)融合所形成的突变蛋白，或(iv)附加的氨基酸序列融合到此突变蛋白序列而形成的突变蛋白(如前导序列或分泌序列或用来纯化此突变蛋白的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。本发明中，保守性替换的氨基酸最好根据表I进行氨基酸替换而产生。
表I
本发明的活性突变蛋白具有顺式切割活性且反式切割活性降低，甚至没有反式切割活性。
优选地，所述的突变蛋白如SEQ ID NO.：2-3中任一所示。
突变体蛋白FnCas12aK1069R氨基酸序列：
突变型蛋白FnCas12aF1081R氨基酸序列：

应理解，本发明突变蛋白与SEQ ID NO.：2-3中任一所示的序列相比，通常具有较高的同源性(相同性)，优选地，所述的突变蛋白与SEQ ID NO.：2-3中任一所示序列的同源性至少为80％，较佳地至少为85％-90％，更佳地至少为95％，最佳地至少为98％或99％。
此外，还可以对本发明突变蛋白进行修饰。修饰(通常不改变一级结构)形式包括：体内或体外的突变蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在突变蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的突变蛋白。这种修饰可以通过将突变蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨 酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的突变蛋白。
术语“编码突变蛋白的多核苷酸”可以是包括编码本发明突变蛋白的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
在一优选实施方式中，本发明的编码突变蛋白的多核苷酸的序列如SEQ ID NO.：4-5中任一所示。
FnCas12aK1069R核苷酸序列(SEQ ID NO.4)：

FnCas12aF1081R核苷酸序列(SEQ ID NO.5)：


本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或突变蛋白的片段、类似物和衍生物。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的突变蛋白的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的突变蛋白和多核苷酸优选以分离的形式提供，更佳地，被纯化至均质。
本发明多核苷酸全长序列通常可以通过PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
应注意，本发明中来源于Francisella tularensis的基因编辑蛋白(FnCas12a)氨基酸序列中的1081位点、1069位点在其余来源的Cas12a中对应的位点均为保守位点，具体对应关系见表II。
表II突变氨基酸对应位点
因此，上述位点的突变对于降低基因编辑脱靶率具有至关重要的作用。
表达载体和宿主细胞
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明突变蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的突变蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明突变蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码突变蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明突变蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞(如大肠杆菌)，或是低等真核细胞，或是高等真核细胞，如酵母细胞、植物细胞或哺乳动物细胞(包括人和非人哺乳动物)。代表性例子有：大肠杆菌、麦胚细胞，昆虫细胞，SF9、Hela、HEK293、CHO、酵母细胞等。在本发明的一个优选实施方式中，选择酵母细胞(如毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母)为宿主细胞。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的主要优点包括：
(1)本发明首次发现一种新的基因编辑蛋白变体，相比野生型基因编辑蛋白，本发明的基因编辑蛋白变体可具有顺式切割活性且反式切割活性降低，甚至没有反式切割活性，并且本发明的基因编辑蛋白变体以及含有本发明的基因编辑蛋白变体的基因编辑系统可显著降低基因编辑脱靶率。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非有特别说明，否则本发明实施例中的试剂和材料均为市售产品。
(一)材料与方法
1、FnCas12a蛋白突变实验
(1)FnCas12a突变体蛋白表达载体的构建
设计包含突变位点的引物(序列见表1)，以野生型FnCas12a表达质粒为模板，用Phanta DNA polymerase扩增出具有目的位点突变的线性片段，用Ezmax(获自安徽吐露港生物科技有限公司)将扩增产物无缝拼接连接为环状表达载体，将反应产物转入DH10B(获自安徽吐露港生物科技有限公司)，并在50μg/mL Kan的LB培养基37℃过夜培养，挑取单克隆在50μg/mL Kan液体LB培养基中摇菌，37℃过夜，提取质粒。将测序正确的质粒保存于-80℃。
表1
(2)FnCas12a突变体蛋白的纯化
将构建好的pET28TEV-FnCas12a质粒转化E.coli BL21(DE3)感受态，在含有50μg/mL卡那(后面简称Kan)抗性的固体LB培养基37℃培养12-14h。挑3个单克隆到50mL Kan抗性的液体LB培养基中，37℃摇床过夜培养后，1％(v/v)转接到1L含有Kan抗性的液体LB培养基，37℃培养至OD600＝0.6-0.8之间，冰浴30min，加入终浓度为0.2-0.5mM的IPTG，16℃，220rpm培养14-16h。16℃，6000rpm离心5min收菌，将菌沉淀称好重量后开始破菌，也可以暂时保存于-80℃。(以下步骤都要在4℃操作)。按蛋白裂解缓冲液/菌重量为5-10mL/g的比例重悬菌沉淀，同时，加入终浓度为1mM PMSF蛋白抑制剂，菌体重悬均匀后，细胞破碎仪高压裂解，将获得的裂解溶液14000rpm离心30min，收集上清。将离心所得蛋白上清液与 Ni-NTA(天地人和生物科技有限公司)混合，4℃慢慢晃动1h，使蛋白与镍柱充分结合，然后将其装载到30mL的柱上，流尽上清液后，用低浓度咪唑的洗杂缓冲液冲洗杂蛋白，在用高浓度咪唑的洗脱缓冲液洗脱目的蛋白，小体积洗脱目的蛋白(具体操作步骤参考Ni-NTA的操作说明书)。浓度为10％(v/v)的SDS-PAGE胶验证目的蛋白的纯度，将较纯的几管目的蛋白合并，透析过夜后用50KDa超滤管浓缩，蛋白纯度如图1所示。等体积的甘油(提前预冷至4℃)与蛋白混合均匀，用Bradford方法测定蛋白浓度，小体积分装保存于-80℃，短期使用可在-20℃保存。
2、靶标dsDNA序列的制备：
以AMED16s-F/R(序列AMED16s-F：5′-gtgaactaagccagtagagc-3′，AMED16s-R：5′-ctttcgctcctcagcgtcag-3′，生工生物工程(上海)股份有限公司合成)为扩增引物，以地中海拟无枝酸菌U32基因组(NCBI登录号：SAMN02603409)为模板进行PCR扩增。靶标dsDNA片段的PCR扩增体系见表2。PCR反应程序为：95℃预变性10min，95℃变性15s，57℃退火15s，72℃延伸30s(1min可扩增2kb)，32个循环，最后，75℃延伸5min。1.5％(w/v)琼脂糖凝胶电泳鉴定片段大小，扩增产物为正确单一的DNA片段，采用柱回收方法回收目的片段，柱回收用Promega公司的Wizard SV Gel and PCR clean-up system试剂盒。
表2 target dsDNA片段的PCR扩增体系
3、顺式切割反应实验：
表3顺式切割反应体系
表4 10xHOLMES buffer成分
crRNA序列：5′-AAUUUCUACUCUUGUAGAUGCCAGGGACGAAGCGCAAGUGACGGAAU-3′，由南京金斯瑞生物科技有限公司合成，HPLC纯化。检测方法如下：37℃反应40min，85℃灭活5min，加入终浓度为1×DNA loading。将全部反应产物上样，2％(w/v)琼脂糖凝胶电泳，140V电泳25min，EB泡染30min，凝胶成像仪照胶，顺式切割产物约为529bp和300bp的DNA片段。另外，Control的实验体系不加入FnCas12a蛋白。实验结果如图2所示。
4、反式切割活性检测实验
表5反式切割反应体系

HOLMES-P(FQ-reporter)，购自安徽吐露港生物科技有限公司，是一端为FAM荧光发光基团修饰另一端为荧光淬灭基团修饰的短单链DNA探针(5′-TTTTTT-3′)。当该短单链DNA片段完整时，该DNA探针不发荧光；而只有当该单链DNA片段被切开后，淬灭基团与荧光基团分开，才能检测到该DNA探针的荧光信号。配好体系后立刻放入实时荧光定量PCR仪器中检测荧光信号，37℃条件下孵育，每隔一分钟采集一次荧光信号，共计采集30次信号(计60min)，其实验结果如图3所示。该体系中除FnCas12a蛋白外，其它成分均先配成混合体系。另外，Control即实验体系不加入靶标dsDNA。
(二)结果与讨论
本发明对FnCas12a结构进行分析，根据晶体结构6i1k显示的结果，与DNA底物相互作用的FnCas12a氨基酸包括：K1069，F1081，F1010，V1285、N1288等，这些氨基酸位点可能与反式切割活性相关，本发明对这些位点进行突变，并对这些蛋白进行顺式和反式切割活性测定，最后获得两个具有顺式切割活性且没有反式切割活性的突变体蛋白，这两个蛋白的突变分别是1081位氨基酸从苯丙氨酸突变为精氨酸(F1081R)和1069位氨基酸从赖氨酸突变为精氨酸(K1069R)，对应的蛋白名称分别为FnCas12aF1081R和FnCas12aK1069R。野生型蛋白(WT)和突变体蛋白(F1081R和K1069R)的纯化结果如图1。顺式切割活性检测结果显示FnCas12aF1081R和FnCas12aK1069R这两个突变体蛋白的顺式活性和FnCas12a无显著差异(图2)。反式切割活性检测结果表明FnCas12aF1081R和FnCas12aK1069R的反式切割活性相较于野生型FnCas12a蛋白的反式切割活性有显著降低(图3)。
综上所述，本发明发现了两个FnCas12a的突变体蛋白，它们的突变位点分别是1081位氨基酸从苯丙氨酸突变为精氨酸(F1081R)和1069位氨基酸从赖氨酸突变为精氨酸(K1069R)，该两种突变体蛋白保留了顺式切割活性，丧失(或显著降低)原有野生型基因编辑的反式切割活性。由于Cas12a野生型蛋白不但 能够特异性切割靶标DNA，还具有对单链状态的DNA具有非特异的反式切割活性，其在基因编辑过程中会引起一定程度的脱靶效应。在本发明通过对野生型基因编辑蛋白进行人工改造的方法将Cas12a的反式切割活性去除(或降低)的同时保留了其顺式切割活性，克服了由基因编辑蛋白的反式切割活性所引起的脱靶问题，从而使Cas12a突变体蛋白在基因编辑方面更有优势。
此外，2类成簇规则间隔短回文重复序列(CRISPR)-Cas系统以单一效应蛋白为特征，可进一步细分为类型II、V和VI等。V型家族的效应蛋白在N端具有多样性，但在C端保留一个统一的RuvC样内切酶结构域。V型系统进一步细分为许多亚型，包括V-A型到V-I型、V-K型、V-U型和CRISPR-Cas8φ(见图5)。Cas12a(V-A型)、Cas12b(V-B型)和Cas12e(V-E型)都属于V型系统，它们在效应蛋白结合gRNA形成二元复合物后，特异性识别富含5′-T的PAM，并促进靶DNA解旋，同时，靶标序列的非靶标链(NTS)发生位移，形成所谓的“R环”结构。RuvC域在PAM远处连续切割NTS和靶标链(TS)，形成一个有5、7或10个NT链5′突出部分的交错切口。Cas12a，Cas12b，和Cas12e，这三种蛋白质都有由α螺旋识别(REC)叶和核酸酶(NUC)叶组成的双叶结构(见图7)。两个叶通过桥螺旋(BH)结构域连接。REC叶包含两个REC结构域(REC1和REC2)，主要帮助调节和稳定形成“R环”后的crRNA靶与DNA杂交。(Tong Baisong等人.The Versatile Type V CRISPR Effectors and Their Application Prospects[J].Frontiers in Cell and Developmental Biology，2021，8：622103-622103.)
根据图6，FnCas12a的1069位点位于RuvC域。V型系统下各Cas蛋白均存在RuvC域(见图5)及相应位点，可以预料，对V型系统下各Cas蛋白中对应于FnCas12a的1069位点的氨基酸残基进行突变，会获得类似的效果；尤其是对结构、功能更为类似，同源性更高的Cas12a、Cas12b、Cas12e中对应于FnCas12a的1069位点的氨基酸残基进行突变，更会获得类似的效果；更尤其是对结构功能又更为类似、同源性又更高的其余来源的Cas12a(见图4，a-e)中对应于FnCas12a的1069位点的氨基酸残基进行突变(见表II)，更会获得类似效果。
同样，根据图6，FnCas12a的1081位点位于RuvC域与NUC域交界处。Cas12a、Cas12b、Cas12e均存在RuvC域及NUC域(见图7)。可以预料，对结构、功能类似，同源性高的Cas12a、Cas12b、Cas12e中对应于FnCas12a的1081位点的氨基酸残基进行突变，会获得类似的效果；更尤其是结构功能更为类似、同源性高的 其余来源的Cas12a(见图4，a-e)中对应于FnCas12a的1081位点的氨基酸残基进行突变(见表II)，更会获得类似效果。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (21)

  1. 一种基因编辑蛋白变体，其特征在于，所述变体为具有顺式切割活性的非天然蛋白，且所述变体相较于其野生型基因编辑蛋白的反式切割活性降低，并且所述变体在野生型基因编辑蛋白的选自下组一个或多个核心氨基酸位点发生突变：
    对应于FnCas12a第1081位的苯丙氨酸(F)位点；和/或
    对应于FnCas12a第1069位的赖氨酸(K)位点。
  2. 如权利要求1所述的基因编辑蛋白变体，其特征在于，FnCas12a第1081位的苯丙氨酸(F)突变为选自下组的一种或多种氨基酸：精氨酸(R)、酪氨酸(Y)、色氨酸(W)、谷氨酰胺(Q)、天冬酰胺(N)、赖氨酸(K)、谷氨酸(E)、天冬氨酸(D)或其组合。
  3. 如权利要求1所述的基因编辑蛋白变体，其特征在于，FnCas12a第1069位的赖氨酸(K)突变为选自下组的一种或多种氨基酸：精氨酸(R)、酪氨酸(Y)、谷氨酰胺(Q)、天冬酰胺(N)、赖氨酸(K)、谷氨酸(E)、天冬氨酸(D)或其组合。
  4. 如权利要求1所述的基因编辑蛋白变体，其特征在于，所述基因编辑蛋白为V型CRISPR/Cas蛋白。
  5. 如权利要求1所述的基因编辑蛋白变体，其特征在于，所述基因编辑蛋白选自下组：Cas 12、Cas14、或其组合。
  6. 如权利要求1所述的基因编辑蛋白变体，其特征在于，所述基因编辑蛋白选自下组：Cas12a、Cas12b、Cas12e或其组合。
  7. 如权利要求1所述的基因编辑蛋白变体，其特征在于，所述基因编辑蛋白为FnCas12a。
  8. 一种多核苷酸，其特征在于，所述的多核苷酸编码权利要求1所述的变体。
  9. 一种载体，其特征在于，所述的载体含有权利要求8所述的多核苷酸。
  10. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求9所述的载体，或其基因组中整合有权利要求8所述的多核苷酸。
  11. 一种基因编辑蛋白变体的制备方法，其特征在于，所述的方法包括步骤：
    (a)在适合表达的条件下，培养权利要求10所述的宿主细胞，从而表达所述的基因编辑蛋白变体；和
    (b)分离所述的基因编辑蛋白变体。
  12. 一种酶制剂，其特征在于，所述酶制剂包括权利要求1所述的基因编辑蛋白变体。
  13. 一种基因编辑系统，其特征在于，包括：
    权利要求1所述的基因编辑蛋白变体、或其编码基因或其表达载体；和
    gRNA或其表达载体，和/或其用于靶标位点断裂修复的寡核苷酸或核酸片段或质粒。
  14. 一种基因编辑试剂，其特征在于，所述基因编辑试剂包含权利要求1所述的基因编辑蛋白变体。
  15. 一种组合物，其特征在于，包括：
    权利要求1所述的基因编辑蛋白变体或权利要求13所述的系统或权利要求14所述的基因编辑试剂；和
    药学上可接受的载体。
  16. 一种产品组合，其特征在于，包括：
    权利要求1所述的基因编辑蛋白变体或权利要求13所述的系统或权利要求14所述的基因编辑试剂。
  17. 一种试剂盒，其特征在于，包括：权利要求1所述的基因编辑蛋白变体或或权利要求12所述的酶制剂或权利要求13所述的基因编辑系统或权利要求14所述的基因编辑试剂或权利要求15所述的组合物或权利要求16所述的产品组合。
  18. 一种药盒，其特征在于，包括：
    第一容器，以及位于所述第一容器中的权利要求1所述的基因编辑蛋白变体或权利要求12所述的酶制剂或权利要求13所述的基因编辑系统或权利要求14所述的基因编辑试剂或权利要求15所述的组合物或权利要求16所述的产品组合，或含有权利要求1所述的基因编辑蛋白变体或权利要求12所述的酶制剂或权利要求13所述的基因编辑系统或权利要求14所述的基因编辑试剂或权利要求15所述的组合物或权利要求16所述的产品组合的药物。
  19. 一种药盒，其特征在于，包括：
    (a1)第一容器，以及位于所述第一容器中的权利要求1所述的基因编辑蛋白变体、或其编码基因或其表达载体，或含有权利要求1所述的基因编辑蛋白变体、或其编码基因或其表达载体的药物；
    (b1)第二容器，以及位于所述第二容器中的gRNA或其表达载体，或含有gRNA或其表达载体的药物。
  20. 一种权利要求1所述的基因编辑蛋白变体、权利要求12所述的酶制剂、权利要求13所述的基因编辑系统、权利要求14所述的基因编辑试剂或权利要求15所述的组合物或权利要求16所述的产品组合或权利要求18或19所述的药盒的用途，其特征在于，用于制备用于降低基因编辑脱靶率的试剂或试剂盒。
  21. 一种降低基因编辑脱靶率的方法，其特征在于，包括步骤：
    在权利要求1所述的基因编辑蛋白变体、权利要求12所述的酶制剂、权利要求13所述的基因编辑系统、权利要求14所述的基因编辑试剂或权利要求15所述的组合物或权利要求16所述的产品组合或权利要求18或19所述的药盒的存在下，对细胞进行基因编辑，从而降低基因编辑脱靶率。
PCT/CN2023/085720 2022-05-10 2023-03-31 可降低基因编辑脱靶率的基因编辑蛋白变体 WO2023216764A1 (zh)

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