Nuclease, gene editing system containing nuclease and application

Abstract

The invention relates to the technical field of biology, in particular to nuclease, a gene editing system containing the nuclease and application of the nuclease. The nuclease disclosed by the invention is mutant AsCas12f1 nuclease, and compared with wild AsCas12f1 nuclease, the nuclease disclosed by the invention comprises one or more point mutations in the 8th site, the 69th site, the 70th site, the 103th site, the 104th site, the 107th site, the 108th site, the 115th site, the 118th site and the 364th site of amino acid. The gene editing system containing the mutant AsCas12f1 nuclease, disclosed by the invention, can be used for realizing precise gene editing in cells; and the method has extremely high editing efficiency and applicability.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a nuclease and a gene editing system and uses thereof. Background Art

[0002] Genome editing technology refers to a genetic engineering technology that uses gene editing machines, such as programmable nucleases (molecular scissors), to interrupt specific gene sequences and then introduce gene insertion, deletion or replacement to achieve the transformation of specific fragments of the organism's genomic DNA, thereby achieving the editing of the target gene.

[0003] Since the advent of the CRISPR / Cas (Clustered regularly interspaced short palindromicrepeats) genome editing system, it has been widely used in basic and applied research in various fields such as biology, medicine, and agriculture due to its simplicity and efficiency. Cas nucleases can use guide RNA to locate specific target sites in the genome of various cells, cut them to produce double-stranded DNA breaks, and then use endogenous or exogenous DNA repair mechanisms of the cell, such as homologous recombination and non-homologous recombination end-joining repair mechanisms to achieve editing. Depending on the activation of different DNA repair pathways, genome editing will lead to gene inactivation or mutation correction. By fusing the inactivated Cas nuclease with base deaminase or reverse transcriptase, the CRISPR / Cas system can be further transformed into a base editing system and a guide editing system, which are widely used in biology, agricultural research and disease treatment.

[0004] The widely used CRISPR / Cas genome editing systems are mainly of two types: CRISPR / Cas9 and CRISPR / Cas12a. In these two types of genome editing systems, the CRISPR effector protein nucleases Cas9 and Cas12a are large proteins containing more than 1,000 amino acids. The gene editors developed with them as the core have large molecular sizes and are difficult to be effectively packaged and delivered by common vectors such as adeno-associated viruses, which leads to huge problems in the delivery of CRISPR / Cas9 and CRISPR / Cas12a to cells. The extremely small nuclease CRISPR-AsCas12f1 gene editing system in the prior art contains only 422 amino acids, and the gene size is less than half of Cas9 and Cas12a. It naturally has gene editing capabilities and can achieve efficient gene editing in bacteria and mammalian cells. Thanks to its compact molecular size, both the CRISPR-AsCas12f1 gene editing system itself and its derived gene editing tools can easily achieve single AAV packaging and delivery. However, the gene editing ability of the natural AsCas12f1 nuclease is still relatively lower than that of the widely used Cas9 and Cas12a nucleases, which limits the application of AsCas12f1 nuclease in the field of gene editing. Summary of the invention

[0005] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a nuclease and a gene editing system and use comprising the same, so as to solve the problem that the gene editing ability of the wild-type AsCas12f1 nuclease in the prior art is weak.

[0006] To achieve the above-mentioned object and other related objects, the present invention provides a mutant AsCas12f1 nuclease, which comprises one or more point mutations in amino acids 8, 69, 70, 103, 104, 107, 108, 115, 118, and 364 relative to the wild-type AsCas12f1 nuclease; the nucleotide sequence encoding the wild-type AsCas12f1 nuclease is shown in SEQ ID No.24.

[0007] Preferably, the point mutation has one or more of the following characteristics:

[0008] 1. The glutamic acid E at position 8 in the wild-type AsCas12f1 nuclease was mutated to arginine R;

[0009] 2. The threonine T at position 69 in the wild-type AsCas12f1 nuclease was mutated to serine S;

[0010] 3. The asparagine N at position 70 in the wild-type AsCas12f1 nuclease was mutated to glutamine Q;

[0011] 4. Lysine K at position 103 in the wild-type AsCas12f1 nuclease was mutated to arginine R;

[0012] 5. The alanine A at position 104 in the wild-type AsCas12f1 nuclease was mutated to arginine R;

[0013] 6. Lysine K at position 107 in the wild-type AsCas12f1 nuclease was mutated to arginine R;

[0014] 7. The glutamic acid E at position 108 in the wild-type AsCas12f1 nuclease was mutated to arginine R;

[0015] 8. The serine S at position 115 in the wild-type AsCas12f1 nuclease was mutated to arginine R;

[0016] 9. The serine S at position 118 in the wild-type AsCas12f1 nuclease was mutated to alanine A;

[0017] 10. Aspartic acid D at position 364 in the wild-type AsCas12f1 nuclease mutated to arginine R.

[0018] The present invention also provides an isolated polynucleotide comprising the nucleotide sequence of the aforementioned mutant AsCas12f1 nuclease.

[0019] The present invention also provides a nucleic acid construct, which comprises the aforementioned polynucleotide.

[0020] The present invention also provides a gene editing system, which comprises the aforementioned mutant AsCas12f1 nuclease and gRNA, the aforementioned polynucleotide and gRNA, or the aforementioned nucleic acid construct.

[0021] The present invention also provides a composition, wherein the gene editing system comprises the aforementioned mutant AsCas12f1 nuclease, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned gene editing system.

[0022] The present invention also provides an in vitro gene editing method, wherein the target gene is contacted with the aforementioned gene editing system to edit the target gene.

[0023] The present invention also provides the use of the aforementioned mutant AsCas12f1 nuclease, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned gene editing system or the aforementioned composition for performing gene editing on a target gene in an in vitro cell or a cell-free environment.

[0024] The present invention also provides a genetically modified cell, wherein the cell is genetically modified by gene editing of an in vitro cell using the aforementioned mutant AsCas12f1 nuclease, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned gene editing system or the aforementioned composition.

[0025] The present invention also provides the use of the aforementioned mutant AsCas12f1 nuclease in preparing any of the following systems:

[0026] 1. Single-base editing system based on mutant AsCas12f1 nuclease and base deaminase;

[0027] 2. Prime editing system based on mutant AsCas12f1 nuclease and reverse transcriptase;

[0028] 3. Transcription activation system based on mutant AsCas12f1 nuclease and transcription activator;

[0029] 4. Epigenetic modification system based on mutant AsCas12f1 nuclease and epigenetic modification system;

[0030] 5. Transcription inhibition system based on inactivated mutant AsCas12f1 nuclease.

[0031] As described above, a nuclease and a gene editing system and use comprising the same of the present invention have the following beneficial effects:

[0032] Compared with the wild-type system, the gene editing system containing the mutant AsCas12f1 nuclease in the present invention has significantly improved the gene editing efficiency of 138 target sites in mammalian cells. The present invention further improves the editing efficiency and applicability of the extremely small CRISPR / Cas12f system in cell gene editing, and can achieve precise gene editing in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shown is a comparison of the mammalian cell gene editing results of the wild-type AsCas12f1 nuclease and the mutant AsCas12f1 nuclease of the present invention.

[0034] Figure 2 Shown is a diagram of the mammalian cell gene editing results of the mutant AsCas12f1 nuclease with combined mutations of the present invention.

[0035] Figure 3 A graph showing a comparison of the gene editing effects of the wild-type CRISPR / AsCas12f1 system and the mutant CRISPR / AsCas12f1 system based on the sgRNA_T1 nuclease in mammalian cells. DETAILED DESCRIPTION

[0036] The present invention provides a mutant AsCas12f1 nuclease, wherein the mutant AsCas12f1 nuclease comprises one or more point mutations in amino acids at positions 8, 69, 70, 103, 104, 107, 108, 115, 118, and 364 relative to the wild-type AsCas12f1 nuclease, that is, the variant AsCas12f1 nuclease may include a point mutation in amino acids at positions 8, 69, 70, 103, 104, 107, 108, 115, 118, and 364, or comprises more than one combined mutation described above. The nucleotide sequence encoding the wild-type AsCas12f1 nuclease is shown in SEQ ID No.24.

[0037] In some specific embodiments, the AsCas12f1 nuclease comprising the point mutation is selected from AsCas12f1-E8R, i.e., the glutamic acid E at position 8 in the wild-type AsCas12f1 nuclease is mutated to arginine R, AsCas12f1-T69S, i.e., the threonine T at position 69 in the wild-type AsCas12f1 nuclease is mutated to serine S, AsCas12f1-N70Q, i.e., the asparagine N at position 70 in the wild-type AsCas12f1 nuclease is mutated to glutamine Q, AsCas12f1-K103R, i.e., the lysine K at position 103 in the wild-type AsCas12f1 nuclease is mutated to arginine R, AsCas12f1-A104R, i.e., the threonine T at position 69 in the wild-type AsCas12f1 nuclease is mutated to serine S, Alanine A is mutated to arginine R, AsCas12f1-K107R, i.e., lysine K at position 107 in the wild-type AsCas12f1 nuclease is mutated to arginine R, AsCas12f1-E108R, i.e., glutamate E at position 108 in the wild-type AsCas12f1 nuclease is mutated to arginine R, AsCas12f1-S115R, i.e., serine S at position 115 in the wild-type AsCas12f1 nuclease is mutated to arginine R, AsCas12f1-S118A, i.e., serine S at position 118 in the wild-type AsCas12f1 nuclease is mutated to alanine A, or AsCas12f1-D364R, i.e., aspartic acid D at position 364 in the wild-type AsCas12f1 nuclease is mutated to arginine R. The nucleotide sequence encoding the wild-type AsCas12f1 nuclease is shown in SEQ ID No.24.

[0038] In some specific embodiments, the mutant AsCas12f1 nuclease comprising the combined mutation is selected from AsCas12f1-N70Q+A104R (referred to as AsCas12f1-v2.1), i.e., the 70th and 104th positions in the wild-type AsCas12f1 nuclease are mutated simultaneously, AsCas12f1-K103R+A104R (referred to as AsCas12f1-v2.2), i.e., the 103rd and 104th positions in the wild-type AsCas12f1 nuclease are mutated simultaneously, AsCas12f1-A104R+K107R (referred to as AsCas12f1-v2.3), i.e., the 104th and 104th positions in the wild-type AsCas12f1 nuclease are mutated simultaneously. The 107th position mutated simultaneously, AsCas12f1-A104R+E108R (referred to as AsCas12f1-v2.4) means that the 104th and 108th positions in the wild-type AsCas12f1 nuclease mutated simultaneously, AsCas12f1-A104R+S115R (referred to as AsCas12f1-v2.5) means that the 104th and 115th positions in the wild-type AsCas12f1 nuclease mutated simultaneously, AsCas12f1-A104R+S118A (referred to as AsCas12f1-v2.6) means that the 104th and 118th positions in the wild-type AsCas12f1 nuclease mutated simultaneously, and AsCas12f1-A104R+D3 64R (referred to as AsCas12f1-v2.7) means that the 104th and 364th positions in the wild-type AsCas12f1 nuclease are mutated simultaneously, AsCas12f1-N70Q+A104R+D364R (referred to as AsCas12f1-v3.1) means that the 70th, 104th and 364th positions in the wild-type AsCas12f1 nuclease are mutated simultaneously, AsCas12f1-N70Q+A104R+S115R+D364R (referred to as AsCas12f1-v4.1) means that the 70th, 104th, 115th and 364th positions in the wild-type AsCas12f1 nuclease are mutated simultaneously, and AsCas12f1-N7 0Q+A104R+S118A+D364R (referred to as AsCas12f1-v4.2), that is, the 70th, 104th, 118th and 364th positions in the wild-type AsCas12f1 nuclease are mutated simultaneously, AsCas12f1-N70Q+K103R+A104R+S118A+D364R (referred to as AsCas12f1-v5.1), that is, the 70th, 103rd, 104th, 118th and 364th positions in the wild-type AsCas12f1 nuclease are mutated simultaneously, and AsCas12f1-N70Q+A104R+K107R+S118A+D364R (referred to as AsCas12f1-v5.2) That is, the 70th, 104th, 107th, 118th and 364th positions in the wild-type AsCas12f1 nuclease are mutated simultaneously, and AsCas12f1-N70Q+K103R+A104R+K107R+S118A+D364R (referred to as AsCas12f1-v6.1) is the 70th, 103rd, 104th, 107th, 118th and 364th positions in the wild-type AsCas12f1 nuclease are mutated simultaneously. Preferably, the AsCas12f1 nuclease containing the combined mutation is AsCas12f1-v5.1. The nucleotide sequence encoding the wild-type AsCas12f1 nuclease is shown in SEQ ID No.24. .

[0039] Furthermore, the mutant AsCas12f1 nuclease comprises an amino acid sequence as shown in any one of SEQ ID No.1-23. wherein the variant AsCas12f1-E8R comprises an amino acid sequence such as SEQ ID No.1; the variant AsCas12f1-T69S comprises an amino acid sequence such as SEQ ID No.2; the variant AsCas12f1-N70Q comprises an amino acid sequence such as SEQ ID No.3; the variant AsCas12f1-K103R comprises an amino acid sequence such as SEQ ID No.4; the variant AsCas12f1-A104R comprises an amino acid sequence such as SEQ ID No.5; the variant AsCas12f1-K107R comprises an amino acid sequence such as SEQ ID No.6; the variant AsCas12f1-E108R comprises an amino acid sequence such as SEQ ID No.7; the variant AsCas12f1-S115R comprises an amino acid sequence such as SEQ ID No.8; the variant AsCas12f1-S118A comprises an amino acid sequence such as SEQ ID No.9; the variant AsCas12f1-D364R comprises an amino acid sequence such as SEQ ID The amino acid sequence of AsCas12f1-A104R+S115R comprises the amino acid sequence of SEQ ID No.15; the variant AsCas12f1-A104R+S118A comprises the amino acid sequence of SEQ ID No.16; the variant AsCas12f1-A104R+D364R comprises the amino acid sequence of SEQ ID No.17. No.17; variant AsCas12f1-N70Q+A104R+D364R comprises an amino acid sequence such as SEQ ID No.18; variant AsCas12f1-N70Q+A104R+S115R+D364R comprises an amino acid sequence such as SEQ ID No.19; variant AsCas12f1-N70Q+A104R+S118A+D364R comprises an amino acid sequence such as SEQ ID No.20; variant AsCas12f1-N70Q+K103R+A104R+S118A+D364R comprises an amino acid sequence such as SEQ ID No.The variant AsCas12f1-N70Q+A104R+K107R+S118A+D364R comprises the amino acid sequence of SEQ ID No. 21; the variant AsCas12f1-N70Q+A104R+K107R+S118A+D364R comprises the amino acid sequence of SEQ ID No. 22; the variant AsCas12f1-N70Q+K103R+A104R+K107R+S118A+D364R comprises the amino acid sequence of SEQ ID No. 23. The variant can be understood as the mutant AsCas12f1 nuclease. .

[0040] The present invention also provides an isolated polynucleotide comprising a nucleotide sequence encoding the mutant AsCas12f1 nuclease.

[0041] In some specific embodiments, the polynucleotide comprises a nucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID No. 1-23.

[0042] In some embodiments, the polynucleotides contain one or more modifications that may be useful, for example, to enhance activity, stability or specificity, to alter delivery, to reduce innate immune responses in host cells, or for other enhancements.

[0043] In some embodiments, the polynucleotide may also be composed of nucleotides and / or nucleotide mimics. Specifically, the polynucleotide is composed of polynucleotide mimics peptide nucleic acids with excellent hybridization properties.

[0044] The present invention also provides a nucleic acid construct, which comprises the aforementioned polynucleotide. The nucleic acid construct can generally be constructed by inserting the isolated polynucleotide into a suitable expression vector, and a person skilled in the art can select a suitable expression vector. The nucleic acid construct can be, for example, a recombinant expression vector, and any suitable expression vector can be used as long as it is compatible with the host cell, including but not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus; adeno-associated virus; SV40; herpes simplex virus; human immunodeficiency virus; retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), etc.

[0045] In some embodiments, the nucleic acid construct further comprises a nucleotide sequence or a regulatory element encoding a gRNA. Specifically, the regulatory element is selected from a promoter, an enhancer, a nuclear localization signal or a poly A structure. More specifically, the promoter is selected from a wild-type promoter, an inducible promoter or a constitutive promoter.

[0046] In some embodiments, the regulatory element can function in eukaryotic cells, such as mammalian cells (HEK293T cells); or prokaryotic cells (such as bacterial or archaeal cells). The regulatory element is connected to the nucleotide sequence encoding the nuclease, which allows the nucleotide sequence encoding the nuclease to be expressed in both prokaryotic and eukaryotic cells.

[0047] The present invention also provides an expression system, the expression system comprising the aforementioned nucleic acid construct or the aforementioned polynucleotide integrated into the genome. The host cell of the expression system is selected from eukaryotic cells or prokaryotic cells; preferably, the host cell is selected from mouse cells and human cells.

[0048] The present invention also provides a gene editing system, which comprises the aforementioned mutant AsCas12f1 nuclease and gRNA or a polynucleotide encoding gRNA; or, comprises the aforementioned polynucleotide and gRNA or a polynucleotide encoding gRNA; or, comprises the aforementioned nucleic acid construct and gRNA or a polynucleotide encoding gRNA; or, comprises the aforementioned nucleic acid construct comprising a nucleotide sequence encoding gRNA.

[0049] In some embodiments, the gene editing system recognizes a PAM sequence on a targeting sequence; preferably, the PAM sequence is 5'-TTR, where R represents A or G. The gene editing system targets a nucleic acid fragment of 12 to 40 bp in length after the PAM sequence, preferably 20 bp in length. The gene editing system targets at least one target sequence in the cell genome.

[0050] In the editing system of the present invention, the aforementioned polynucleotide includes: a coding sequence encoding only a nuclease; a coding sequence of a nuclease and various additional coding sequences; a coding sequence of a nuclease (and optional additional coding sequences) and a non-coding sequence. In some embodiments, the gene editing system comprises one or more vectors; the one or more vectors comprise (i) a first regulatory element, the first regulatory element being connected to the aforementioned polynucleotide; and (ii) a second regulatory element, the second regulatory element being connected to a polynucleotide encoding a guide RNA nucleotide sequence, i.e., a gRNA sequence; the (i) and (ii) are located on the same or different vectors. In some embodiments, the gene editing system comprises (i) a nuclease or a variant thereof, and (ii) a vector comprising a nucleotide sequence encoding the guide RNA. In another embodiment, the system comprises a gRNA and a nuclease complex.

[0051] The first regulatory element can regulate the transcription of the polynucleotide encoding the nuclease or its variant. The polynucleotide encoding the nuclease or its variant can be one or more, and the first regulatory element can be one or more. The second regulatory element can regulate the transcription of the polynucleotide encoding the guide RNA. The polynucleotide encoding the guide RNA can be one or more, and the second regulatory element can be one or more. The first regulatory element or the second regulatory element is selected from a promoter, an enhancer, a nuclear localization signal or a poly A structure.

[0052] The system of the present invention can include one gRNA or multiple gRNAs at the same time. In one embodiment, the system includes multiple gRNAs at the same time to modify different positions on the same target DNA or different target DNAs at the same time. In one embodiment, two or more guide RNAs target the same gene or transcript or locus. In one embodiment, two or more guide RNAs target different unrelated loci. In some embodiments, two or more guide RNAs target different but related loci.

[0053] In the present invention, the polynucleotide or the nucleic acid construct can be synthesized by artificial synthesis, such as by chemical synthesis, so that it is easy to carry out a variety of modifications. The modification can be any modification known in the art. In some embodiments, the polynucleotide or the nucleic acid construct comprises one or more modifications, so that it is easy to incorporate many modifications, such as enhancing transcriptional activity, changing enzyme activity, improving its translation or stability (such as increasing its resistance to proteolysis, degradation) or specificity, changing solubility, changing delivery, reducing the innate immune response in the host cell. The modification can be any modification known in the art. In some embodiments, the DNA or RNA encoding the nuclease introduced into the cell by modification is used to edit any one or more genomic loci. The modification can be a single modification, or a combination of modifications.

[0054] In the present invention, the mutant AsCas12f1 nuclease, or the polynucleotide encoding the mutant AsCas12f1 nuclease is applicable to any biological or in vitro environment, including but not limited to bacteria (such as Escherichia coli, Klebsiella pneumoniae), archaea, fungi, protists, plants or animals. Accordingly, applicable target cells include but are not limited to eukaryotic cells and prokaryotic cells, such as bacterial cells, archaeal cells, fungal cells, protist cells, plant cells or animal cells; the eukaryotic cells include mammalian cells and plant cells, and the prokaryotic cells include Escherichia coli and Klebsiella pneumoniae. Applicable target cells can be any type of cell, including stem cells, somatic cells, etc. The present invention is preferably used for mammalian cell HEK293T cells. The cell can be in vivo or in vitro. In some embodiments, the variant AsCas12f1 nuclease and gRNA, the polynucleotide and gRNA or the nucleic acid construct are formulated in liposomes or lipid nanoparticles.

[0055] In the gene editing system of the present invention, the nuclease and gRNA can form a complex in the host cell to recognize the PAM sequence on the targeted gene (such as targeted DNA) sequence; the targeting sequence of the CRISPR / AsCas12f1 gene editing system is a nucleic acid fragment (such as a DNA fragment) with a length of 20bp after the PAM sequence. In one embodiment, the complex can selectively regulate the transcription of the target DNA in the host cell. The gene editing system can cut the double strand of the targeted DNA, causing DNA breaks.

[0056] In the present invention, nuclease variants can also be formed by modification, mutation, DNA reorganization, etc., so that the nuclease variant has improved desired characteristics, such as function, activity, kinetics, half-life, etc. The modification can be, for example, the deletion, insertion or substitution of amino acids, and another example can be the replacement of the "cleavage domain" of nucleases such as AsCas12f1 with homologous or heterologous cleavage domains from different nucleases (e.g., the HNH domain of CRISPR-associated nucleases); by any modification method of DNA binding and / or DNA modification proteins known in the art, such as methylation, demethylation, acetylation, etc., for example, the DNA targeting of AsCas12f1 nuclease can be changed. The DNA reorganization refers to the exchange of sequence fragments between DNA sequences of AsCas12f1 nucleases from different sources to produce a chimeric DNA sequence encoding a synthetic protein with RNA-guided endonuclease activity. The modification, mutation, DNA reorganization, etc. can be used alone or in combination.

[0057] The present invention also provides a composition, which comprises one or more of the aforementioned mutant AsCas12f1 nuclease, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned gene editing system. In some specific embodiments, the composition further comprises a pharmaceutically acceptable carrier, medium, etc. The acceptable carrier, medium such as sterile water or saline, stabilizer, excipient, antioxidant (ascorbic acid, etc.), buffer (phosphoric acid, citric acid, other organic acids, etc.), preservative, surfactant (PEG, Tween, etc.), chelating agent (EDTA, etc.), adhesive, etc. In addition, other low molecular weight polypeptides may also be contained; proteins such as serum albumin, gelatin or immunoglobulin; amino acids such as glycine, glutamine, asparagine, arginine and lysine; sugars or carbohydrates such as polysaccharides and monosaccharides; sugar alcohols such as mannitol or sorbitol. When preparing an aqueous solution for injection, such as physiological saline, isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, appropriate solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), non-ionic surfactants (Tween 80, HCO-50), etc. can be used. In some embodiments, the composition comprises gRNA and a buffer for stabilizing nucleic acids.

[0058] The present invention also provides a kit comprising one or more of the aforementioned mutant AsCas12f1 nuclease, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned gene editing system or the aforementioned composition. In some specific embodiments, the kit further comprises a dilution buffer, a washing buffer or a control reagent.

[0059] The present invention also provides an in vitro gene editing method, which contacts the target gene with the aforementioned gene editing system to achieve editing of the target gene.

[0060] In some embodiments, the gene editing method comprises the following steps:

[0061] a. introducing the aforementioned mutant AsCas12f1 nuclease, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned gene editing system into an in vitro cell or a cell-free system;

[0062] b. introducing gRNA, also known as sgRNA, or a nucleic acid (e.g., DNA) suitable for in situ production of such gRNA;

[0063] c. contacting a cell or a target gene with the aforementioned mutant AsCas12f1 nuclease, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned gene editing system, a gRNA (sgRNA) or a nucleic acid suitable for in situ production of such a gRNA to produce one or more cuts, incisions or edits in the target gene; wherein the mutant AsCas12f1 nuclease is guided to the target gene by its processed or unprocessed form of the gRNA.

[0064] The present invention also provides the use of the aforementioned mutant AsCas12f1 nuclease, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned gene editing system, the aforementioned composition or the aforementioned kit for performing gene editing on a target gene in an in vitro cell or a cell-free environment.

[0065] In some specific embodiments, the in vitro cell is selected from one or more of bacterial cells, archaeal cells, fungal cells, protist cells, viral cells, plant cells and animal cells.

[0066] In some embodiments, the function of gene editing is selected from one or more of gene cutting, gene deletion, gene insertion, point mutation, transcription inhibition or transcription activation. More specifically, the function of gene editing is selected from: cutting the target gene;

[0067] Manipulate the expression of target genes;

[0068] Genetic modification of target genes;

[0069] Genetically modified target gene-related polypeptides;

[0070] For intentional and controlled lesions at any desired location in a target gene;

[0071] For deliberate and controlled repair at any desired location in a target gene;

[0072] The target gene is modified by a method other than introducing a double-strand break (the AsCas12f1 nuclease has enzymatic activity, which modifies the target gene in a method other than introducing a double-strand break; the enzymatic activity may be possessed by AsCas12f1 itself, or obtained by, for example, fusing a heterologous polypeptide having enzymatic activity to the AsCas12f1 nuclease to form a chimeric AsCas12f1 nuclease, and the enzymatic activity includes but is not limited to methyltransferase activity, deamination activity, dismutase activity, alkylation activity, demethylase activity, DNA repair activity, transposase activity, recombinase activity, DNA damage activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, etc.).

[0073] In some specific embodiments, the target gene is a target DNA. In some embodiments, the target DNA may be an in vitro naked DNA that is not bound to a DNA-related protein. In some embodiments, the target DNA is a chromosomal DNA in an in vitro cell. In some embodiments, the target gene is a target RNA. In some embodiments, the target DNA is contacted with a targeting complex comprising the mutant AsCas12f1 nuclease and a gRNA, and the gRNA provides target specificity for the targeting complex by comprising a nucleotide sequence complementary to the target DNA; the AsCas12f1 nuclease provides site-specific activity. In some embodiments, the targeting complex modifies the target DNA, resulting in, for example, DNA cutting, DNA methylation, DNA damage, DNA repair, and the like. In some embodiments, the targeting complex modifies polypeptides associated with the target DNA (e.g., histones, DNA-binding proteins, etc.), resulting in, for example, methylation of target DNA-associated polypeptides-histones, histone acetylation, histone ubiquitination, and the like.

[0074] In the method of the present invention, AsCas12f1 nuclease or a nucleic acid comprising a nucleotide sequence of a polypeptide encoding AsCas12f1 nuclease can be introduced into a cell by a known method. Known methods include DEAE-dextran-mediated transfection, liposome-mediated transfection, virus or phage infection, lipofection, transfection, conjugation, protoplast fusion, polyethyleneimine-mediated transfection, electroporation, calcium phosphate precipitation, gene gun, calcium phosphate precipitation, microinjection, nanoparticle-mediated nucleic acid delivery, etc. For example, plasmids are delivered by electroporation, calcium chloride transfection, microinjection, and lipofection. For viral vector delivery, cells are contacted with viral particles comprising nucleic acids encoding gRNA and / or AsCas12f1 nuclease and / or chimeric AsCas12f1 nuclease and / or donor polynucleotides.

[0075] In some embodiments, in the uses or methods described herein, the nuclease cleaves the target DNA in the cell to generate a double-strand break, which is then repaired by the cell, typically by non-homologous end joining (NHEJ) and homology-directed repair.

[0076] The present invention also provides a genetically modified cell, comprising genetically modifying an in vitro cell by gene editing using the aforementioned mutant AsCas12f1 nuclease, the aforementioned polynucleotide, the aforementioned nucleotide construct, the aforementioned gene editing system, and the aforementioned composition.

[0077] The present invention also provides the use of the aforementioned mutant AsCas12f1 nuclease in preparing any of the following systems:

[0078] 1. Single-base editing system based on mutant AsCas12f1 nuclease and base deaminase;

[0079] 2. Prime editing system based on mutant AsCas12f1 nuclease and reverse transcriptase;

[0080] 3. Transcription activation system based on mutant AsCas12f1 nuclease and transcription activator;

[0081] 4. Epigenetic modification system based on mutant AsCas12f1 nuclease and epigenetic modification system;

[0082] 5. Transcription inhibition system based on inactivated mutant AsCas12f1 nuclease.

[0083] In the present invention, the effective dosage of the mutant AsCas12f1 nuclease and / or the recombinant expression vector and / or the donor polynucleotide is conventional for those skilled in the art and can be determined according to different administration routes and the characteristics of the disease to be treated.

[0084] In the present invention, the bacteria or prokaryotic bacteria can be Escherichia coli, Klebsiella pneumoniae, Bacteroides ovale, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgaris, Bacteroides monomorpha, Lactobacillus casei, Bacteroides fragilis, Acinetobacter lwoffii, Fusobacterium nucleatum, Bacteroides johnsonii, Bacteroides thaliana, Lactobacillus rhamnosus, Bacteroides massiliense, Bacteroides faecalis, Fusobacterium mortis and Bifidobacterium breve, etc.

[0085] In the present invention, the eukaryotic cells include but are not limited to mammalian cells, fungi and other eukaryotic cells. The fungi include yeast and Aspergillus, such as Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyveromyces fragilis, Kluyveromyces lactis, and Schizosaccharomyces pombe, Candida albicans, Candida dunali, Candida glabrata, Candida guilliermondii, Candida kefir, Candida krusei, Candida portugalensis, Candida merlinii, Candida oleophilus, Candida parapsilosis, Candida tropicalis and Candida utilis, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus clavatus, Aspergillus glaucus group, Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus pyrophorus and Aspergillus versicolor.

[0086] The terms "AsCas12f1", "AsCas12f1 nuclease", "AsCas12f1 polypeptide", "AsCas12f1 protein", and "AsCas12f1 protein" are used interchangeably.

[0087] The term "a" or "an" entity refers to one or more of that entity; thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0088] The term "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing corresponding positions in different polypeptides or nucleic acid molecules. When the same position in the sequence of the compared molecules is occupied by the same base or amino acid in the different sequences, then the molecules are homologous at that position. The degree of homology between sequences is determined as a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence should have less than 20% homology to one of the sequences disclosed in the present invention.

[0089] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage of sequence homology (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%) with another polynucleotide or polynucleotide region (or polypeptide or polypeptide region) means that when compared, the percentage of bases (or amino acids) in the two compared sequences are the same. The comparison and percentage homology or sequence identity can be determined using software programs and methods known in the art.

[0090] In the present invention, the terms "polynucleotide" and "oligonucleotide" are used interchangeably, and they refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function known or unknown. Examples of polynucleotides include, but are not limited to, the following: genes or gene fragments (including probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides also include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If there is a modification on the polynucleotide, the modification can be given before or after assembling the polynucleotide. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by coupling to be labeled with a labeling component. The term refers to both double-stranded and single-stranded polynucleotide molecules. Unless otherwise specified or required, any embodiment of a polynucleotide disclosed herein includes a double-stranded form thereof and any of two complementary single-stranded forms known or predicted to be capable of forming a double-stranded form.

[0091] When applied to a polynucleotide, the term "encoding" means that the polynucleotide "encodes" a polypeptide, meaning that it can be transcribed and / or translated to produce a polypeptide of interest and / or fragments thereof, or to produce mRNA capable of encoding the polypeptide of interest and / or fragments thereof, in its native state or when manipulated by methods known to those skilled in the art. The antisense strand refers to a sequence that is complementary to the polynucleotide and from which a coding sequence can be deduced.

[0092] The term "genomic DNA" refers to the DNA of the genome of an organism, including the DNA of the genome of bacteria, archaea, fungi, protists, viruses, plants or animals.

[0093] The term "manipulation" of DNA includes binding, nicking one strand, or cutting both strands of DNA, or includes modifying or editing DNA or a polypeptide bound to DNA. Manipulation of DNA can silence, activate, or regulate the expression of RNA or polypeptides encoded by the DNA (so that it is not transcribed, or the transcriptional activity is reduced, or so that it is not translated, or the translation level is reduced), or prevent or enhance the binding of polypeptides to DNA. Cutting can be performed by a variety of methods, such as enzymatic or chemical hydrolysis of phosphodiester bonds; single-stranded or double-stranded cutting can be performed; DNA cutting can result in the production of blunt ends or staggered ends.

[0094] The term "hybridizable" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA) comprises a nucleotide sequence that enables it to non-covalently bind to another nucleic acid in a sequence-specific, antiparallel manner, i.e., to form Watson-Crick base pairs and / or G / U base pairs, "anneal" or "hybridize" under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength.

[0095] It is understood in the art that the sequence of a polynucleotide need not be 100% complementary to the sequence of a target nucleic acid to which it can specifically hybridize. A polynucleotide may hybridize over one or more segments. A polynucleotide may comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity to a target region within a target nucleic acid sequence to which it is targeted.

[0096] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0097] The term "DNA sequence encoding" a specific RNA is a DNA nucleic acid sequence transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into a protein, or a DNA polynucleotide may encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or gRNA; also referred to as "non-coding" RNA or "ncRNA"). A "protein coding sequence" or a sequence encoding a specific protein or polypeptide is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo or in vitro under the control of a suitable regulatory sequence.

[0098] The term "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus or cosmid, to which another DNA segment, ie, an "insert", can be attached so as to achieve replication of the attached segment in a cell.

[0099] The term "expression cassette" comprises a DNA coding sequence operably linked to a promoter. "Operably linked" means juxtaposed, with the components being in a relationship that allows them to function in their intended manner. The terms "recombinant expression vector" or "DNA construct" are used interchangeably in the present invention to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically produced for the purpose of expressing and / or amplifying inserts or for the construction of other recombinant nucleotide sequences.

[0100] A cell has been "genetically modified" or "transformed" or "transfected" when exogenous DNA, such as a recombinant expression vector, has been introduced into the cell. The presence of the exogenous DNA results in permanent or transient genetic changes. The transforming DNA may or may not integrate into the genome of the cell.

[0101] The term "target DNA" is a DNA polynucleotide comprising a "target site" or "target sequence". The terms "target site", "target sequence", "target protospacer DNA" or "protospacer-like sequence" are used interchangeably in the present invention to refer to a nucleic acid sequence present in the target DNA, to which the DNA-targeting segment of the gRNA will bind if there are sufficient conditions for binding. The RNA molecule contains a sequence that binds, hybridizes or is complementary to the target sequence within the target DNA, thereby targeting the bound polypeptide to a specific position (target sequence) within the target DNA. "Cut" refers to the breakage of the covalent backbone of a DNA molecule.

[0102] The terms "nuclease" and "endonuclease" are used interchangeably to refer to enzymes having endonucleolytic degradation catalytic activity for polynucleotide cleavage. A "cleavage domain" or "active domain" or "nuclease domain" of a nuclease refers to a polypeptide sequence or domain within a nuclease that has catalytic activity for DNA cleavage. The cleavage domain may be contained in a single polypeptide chain, or the cleavage activity may result from the association of two or more polypeptides.

[0103] The term "targeting polypeptide" or "RNA-binding site-guided polypeptide" refers to a polypeptide that binds RNA and targets to a specific DNA sequence.

[0104] The term "guide sequence" or DNA-targeting segment (or "DNA-targeting sequence") comprises a nucleotide sequence (the complementary strand of the target DNA) that is complementary to a specific sequence within the target DNA referred to as a "protospacer-like" sequence in the present invention.

[0105] The term "recombination" refers to the process of exchanging genetic information between two polynucleotides. "Homology-directed repair (HDR)" used in the present invention refers to a specialized form of DNA repair that occurs, for example, during double-strand break repair in a cell. This process requires nucleotide sequence homology, uses a "donor" molecule to provide a template for the repair of a "target" molecule (i.e., a molecule that undergoes a double-strand break), and causes genetic information to be transferred from the donor to the target. If the donor polynucleotide is different from the target molecule, and part or all of the sequence of the donor polynucleotide is incorporated into the target DNA, homology-directed repair may result in changes (e.g., insertions, deletions, mutations) in the sequence of the target molecule.

[0106] The term "non-homologous end joining (NHEJ)" refers to the repair of double-strand breaks in DNA by directly joining the broken ends to each other without the need for a homologous template. NHEJ often results in the deletion of nucleotide sequences near the double-strand break site.

[0107] The term "treating" includes preventing the occurrence of a disease or symptom; inhibiting a disease or symptom or relieving a disease.

[0108] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment or therapy is desired.

[0109] If no specific techniques or conditions are specified in the examples, the conventional techniques or conditions described in the literature in this field or the instructions of the product manufacturer are used. The primers used in the examples were synthesized by Shanghai Sangon Biotechnology Co., Ltd. and Suzhou Genewise Biotechnology Co., Ltd. If the manufacturer of the reagents or instruments is not specified, they are regarded as conventional products that can be purchased through regular channels.

[0110] The CRISPR system mainly used in the present invention is a VF type CRISPR / Cas12f system, in which the effector protein is mainly the original wild Acidibacillus sulfuroxidans Cas12f1 (AsCas12f1) nuclease and its various variants, and the guide RNA is mainly the original wild sgRNA_V1 and the variant sgRNA_T1. AsCas12f1 nuclease or its variants can accurately locate the target gene under the guidance of the corresponding guide RNA or variant, cut the genomic DNA, and achieve double-strand breaks of the genomic DNA. Using the host cell's own or exogenous repair mechanism, the system can efficiently and accurately achieve gene editing in living cells.

[0111] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0112] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0113] When the embodiments give numerical ranges, it should be understood that unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the prior art mastery of those skilled in the art and the records of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention. The variant amino acid sequences in this application are as follows:

[0114] SEQ ID NO.1:

[0115] MIKVYRYRIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0116] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKAYQKEILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0117] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0118] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0119] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0120] YKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.2:

[0121] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0122] SNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKAYQKEILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0123] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0124] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0125] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0126] YKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.3

[0127] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0128] TQVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKAYQKEILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0129] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0130] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0131] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0132] YKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.4

[0133] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0134] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFRAYQKEILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0135] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0136] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0137] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0138] YKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.5

[0139] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0140] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKRYQKEILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0141] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0142] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0143] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0144] YKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.6

[0145] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0146] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKAYQREILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0147] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0148] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0149] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0150] YKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.7

[0151] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0152] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKAYQKRILRGDMSIPSYKRDIPLDLIKENISVNRMN

[0153] HGDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFE

[0154] PQTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGH

[0155] GRDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKI

[0156] IYKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.8

[0157] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0158] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKAYQKEILRGDMRIPSYKRDIPLDLIKENISVNRMN

[0159] HGDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFE

[0160] PQTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGH

[0161] GRDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKI

[0162] IYKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.9

[0163] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0164] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKAYQKEILRGDMSIPAYKRDIPLDLIKENISVNRMN

[0165] HGDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFE

[0166] PQTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGH

[0167] GRDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKI

[0168] IYKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.10

[0169] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0170] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKAYQKEILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0171] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0172] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0173] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0174] YKAEEAGIKVIKIRPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.11

[0175] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0176] TQVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKRYQKEILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0177] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0178] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0179] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0180] YKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.12

[0181] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0182] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFRRYQKEILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0183] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0184] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0185] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0186] YKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.13

[0187] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0188] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKRYQREILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0189] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0190] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0191] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0192] YKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.14

[0193] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0194] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKRYQKRILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0195] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0196] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0197] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0198] YKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.15

[0199] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0200] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKRYQKEILRGDMRIPSYKRDIPLDLIKENISVNRMN

[0201] HGDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFE

[0202] PQTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGH

[0203] GRDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKI

[0204] IYKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.16

[0205] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0206] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKRYQKEILRGDMSIPAYKRDIPLDLIKENISVNRMN

[0207] HGDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFE

[0208] PQTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGH

[0209] GRDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKI

[0210] IYKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.17

[0211] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0212] TNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKRYQKEILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0213] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0214] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0215] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0216] YKAEEAGIKVIKIRPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.18

[0217] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0218] TQVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKRYQKEILRGDMSIPSYKRDIPLDLIKENISVNRMNH

[0219] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0220] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHG

[0221] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0222] YKAEEAGIKVIKIRPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.19

[0223] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0224] TQVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKRYQKEILRGDMRIPSYKRDIPLDLIKENISVNRMN

[0225] HGDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFE

[0226] PQTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGH

[0227] GRDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKI

[0228] IYKAEEAGIKVIKIRPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.20

[0229] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0230] TQVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKRYQKEILRGDMSIPAYKRDIPLDLIKENISVNRMN

[0231] HGDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFE

[0232] PQTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGH

[0233] GRDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKI

[0234] IYKAEEAGIKVIKIRPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.21

[0235] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0236] TQVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFRRYQKEILRGDMSIPAYKRDIPLDLIKENISVNRMN

[0237] HGDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFE

[0238] PQTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGH

[0239] GRDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKI

[0240] IYKAEEAGIKVIKIRPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.22

[0241] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0242] TQVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKRYQREILRGDMSIPAYKRDIPLDLIKENISVNRMN

[0243] HGDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFE

[0244] PQTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGH

[0245] GRDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKI

[0246] IYKAEEAGIKVIKIRPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIKSEQ ID NO.23

[0247] MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGY

[0248] TQVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFRRYQREILRGDMSIPAYKRDIPLDLIKENISVNRMNH

[0249] GDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP

[0250] QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGGG

[0251] RDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKII

[0252] Example 1 Comparison of mammalian cell gene editing results between wild-type AsCas12f1 nuclease and mutant AsCas12f1 nuclease

[0253] The wild-type AsCas12f1 described in this example uses a preferred AsCas12f1 encoding gene optimized for human codons, and its sequence is shown in SEQ ID No. 24:

[0254] SEQ ID No.24

[0255]

[0256] The nucleotide sequence of the plasmid pCMV-AsCas12f1-T1 plasmid sequence carrying wild-type AsCas12f1 is shown in SEQ ID No. 25:

[0257] SEQ ID No.25:

[0258]

[0259] caccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgacgcccgccccacgaccc

[0260] gcagcgcccgaccgaaaggagcgcacgaccccatgcatcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttcta

[0261] gagtcggggcggccggccgcttcgagcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgcttt

[0262] atttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagg

[0263] gggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtaaaatcgataaggatccgtcgaccgatgcccttgagagccttcaa

[0264] cccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggc

[0265] agcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttat

[0266] ccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgt

[0267] ttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccagg

[0268] cgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgct

[0269] ttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctg

[0270] cgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcg

[0271] aggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagcca

[0272] gttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgc

[0273] agaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagat

[0274] tatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgc

[0275] ttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggctt

[0276] accatctggccccagtgctgcaatgataccgcgggacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccg

[0277] agcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcg

[0278] caacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacat

[0279] gatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggc

[0280] agcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcg

[0281] accgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggc

[0282] gaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgttt

[0283] ctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatat

[0284] tattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccga

[0285] aaagtgccacctgcaggaaacagctatgaccatgattacgccaagctctagctagaggtcgacggtatcgagccccagctggttctttccgcctc

[0286] agaagccatagagcccaccgcatccccagcatgcctgctattgtcttcccaatcctcccccttgctgtcctgccccaccccaccccccagaatag

[0287] aatgacacctactcagacaatgcgatgcaatttcctcattttattaggaaaggacagtgggagtggcaccttccagggtcaaggaaggcacggg

[0288] ggaggggcaaacaacagatggctggcaactagaaggcacagtcgaggctgatcagcgggtttaaactcaatggtgatggtgatgatgaccgg

[0289] ttagactttcctcttcttcttgggagaaccaccagacttaatgctctcggcgatgatcttgtcgatgttggggatggcgatattccgggcggcgttgt

[0290] agtcggcgttggcctcgtagccgcaggcccggcacttaaagatggcctggccgattctgttgccggagtcgatgttgccgcactcggagcatct

[0291] ctggctggtgtactgggggtcgatcttgatcactttgatgccggcctcctcggccttgtagatgatcttctgctgcaggtcgtagtaggtccagttct

[0292] gcaggaatctgctgccgatgtctctgatgtttgtcagatcctccatctggattgtgccgcagccctccttgatggccatgtccacgatgtatctgctg

[0293] taccggtgattggtggtgtctctgaaattggcgatcttatccctcagctgctcaatgggcttgattctcttgtctctgccgtggccgcccctggcgcc

[0294] gccggcgtacttgccctgtctcagcatggagatgcgccggctctccacctgcctcctgaagttctcaatctcgccgccctccagcttgtacctggc

[0295] gggggtgtgctggaaggccatgtacacggccacggccacgcccaggtcaatgcccatgatcttgttcaggtccagcactctggtctgtggctcg

[0296] aagtcgtagctgatgttcaggtaccacttgttcttccggtcgtcgtggataatctggctggcgctcacctggtactcgccggacaggattctgtcca

[0297] tgatggtcttgccggcgcccctcacgatgatgatcacggagatctttctcttcacgttcatctcctgcttggcggggttgctcagcagagacaggct

[0298] ggcgatgtagtcgccgtgattcatcctgttcacggagatgttctccttgatcaggtccagggggatgtccctcttgtagctggggatagacatgtc

[0299] gccgcgcaggatctccttctggtaggccttgaacctgtctgtggctctcttgatggtctggctcagatttccagagttcagtctgtaggcctttgtctt

[0300] gatggtgtggtaggcgtagccgtgcacgttggtgtagcccaggatgtccttgctcttggggtactcgccgtggttatccttgtagtcgctgctgaa

[0301] gcccatccactcccaggccagctgtgtggccttattcagggcgaatctggtttcctgctgcagctgtctcaggatggtgccgaactccttccagtc

[0302] caggtccagaggcttcacgatctcgtatctgtacaccttgatcatgactttcctcttcttcttgggGGCcatggtggcggctctccctatagtgagt

[0303] cgtattagcggccgcggatctctagcggatctgacggttcactaaaccagctctgcttatatagacctcccaccgtacacgcctaccgcccatttg

[0304] cgtcaatggggcggagttgttacgacattttggaaagtcccgttgattttggtgccaaaacaaactcccattgacgtcaatggggtggagacttgg

[0305] aaatccccgtgagtcaaaccgctatccacgcccattgatgtactgccaaaaccgcatcaccatggtaatagcgatgactaatacgtagatgtactg

[0306] ccaagtaggaaagtcccataaggtcatgtactgggcataatgccaggcgggccatttaccgtcattgacgtcaatagggggcgtacttggcatat

[0307] gatacacttgatgtactgccaagtgggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccctattggcgttactatgggaacata

[0308] cgtcattattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggccatttaccgtaagttatgtaacgcggaactccatatatgggc

[0309] tatgaactaatgaccccgtaattgattactattaataactagtcaataatcaatgtcaacgcgtatatctggcccgtacatcgcgaagcagcgcaaa

[0310] acgcctaaccctaagcagattcttcatgcaattgtcggtcaagaatcgatagtactaacatacgctctccatcaaaacaaaacgaaacaaaacaaactagcaaaataggctgtccccagtgcaagtgcaggtgccagaacatttctctatcgata-3'

[0311] Using the pCMV-AsCas12f1-T1 plasmid as a template, polymerase chain reaction was performed using point mutation primers targeting different amino acid residues to obtain 10 AsCas12f1 nuclease variant plasmids with one amino acid residue point mutation, which can be used for mammalian cell gene editing.

[0312] The primers used to construct the nuclease single mutant plasmid, i.e., the nucleotide sequence encoding the mutant AsCas12f1 nuclease, comprise the following sequence:

[0313] SEQ ID No.26:E8R-Fw:5'-gcgggtctcacAGAatcgtgaagcctctggacc-3'

[0314] SEQ ID No.27:E8R-Rv:5'-gcgggtctcaTCTgtatctgtacaccttgatcatgac-3'

[0315] SEQ ID No.28:T69S-Fw:5'-gcgggtctcacAGCaacgtgcacggctac-3'

[0316] SEQ ID No.29:T69S-Rv:5'-gcgggtctcaGCTgtagcccaggatgtccttgc-3'

[0317] SEQ ID No.30:N70Q-Fw:5'-gcgggtctcaCTGggtgtagcccaggatgtc-3'

[0318] SEQ ID No.31:N70Q-Rv:5'-gcgggtctcacCAGgtgcacggctacgc-3'

[0319] SEQ ID No.32:K103R-Fw:5'-gcgggtctcacAGAgcctaccagaaggagatcct-3'

[0320] SEQ ID No.33:K103R-Rv:5'-gcgggtctcaTCTgaacctgtctgtggctctcttgat-3'

[0321] SEQ ID No.34:A104R-Fw:5'-gcgggtctcagAGAtaccagaaggagatcctgcgc-3'

[0322] SEQ ID No.35:A104R-Rv:5'-gcgggtctcaTCTcttgaacctgtctgtggctctct-3'

[0323] SEQ ID No.36:K107R-Fw:5'-gcgggtctcagAGAgagatcctgcgcggcga-3'

[0324] SEQ ID No.37:K107R-Rv:5'-gcgggtctcaTCTctggtaggccttgaacctgtct-3'

[0325] SEQ ID No.38:E108R-Fw:5'-gcgggtctcagAGAatcctgcgcggcgacat-3'

[0326] SEQ ID No.39:E108R-Rv:5'-gcgggtctcaTCTcttctggtaggccttgaacctg-3'

[0327] SEQ ID No.40:S115R-Fw:5'-gcgggtctcagAGAatccccagctacaagagggac-3'

[0328] SEQ ID No.41:S115R-Rv:5'-gcgggtctcaTCTcatgtcgccgcgcaggat-3'

[0329] SEQ ID No.42:S118A-Fw:5'-gcgggtctcacGCCtacaagagggacatccccct-3'

[0330] SEQ ID No.43:S118A-Rv:5'-gcgggtctcaGGCggggatagacatgtcgcc-3'

[0331] SEQ ID No.44:D364R-Fw:5'-gcgggtctcacAGAccccagtacaccagccaga-3'

[0332] SEQ ID No.45:D364R-Rv:5'-gcgggtctcaTCTgatcttgatcactttgatgccggc-3'

[0333] The PCR product was digested with Dpn1 and then recovered from the solution. After being assembled by the Golden gate assembly technology, it was transformed into Escherichia coli DH5α competent cells, coated on LBA plates with ampicillin, and a single clone was picked for expansion culture. The plasmid was extracted and sequenced to obtain the pCMV-AsCas12f1-T1 mammalian cell gene editing plasmid encoding the nuclease variants AsCas12f1-E8R, AsCas12f1-T69S, AsCas12f1-N70Q, AsCas12f1-K103R, AsCas12f1-A104R, AsCas12f1-K107R, AsCas12f1-E108R, AsCas12f1-S115R, AsCas12f1-S118A, and AsCas12f1-D364R.

[0334] The variant AsCas12f1-E8R amino acid sequence preferably comprises the sequence shown below: SEQ ID No.1

[0335] The variant AsCas12f1-T69S amino acid sequence preferably comprises the sequence shown below: SEQ ID No.2

[0336] The variant AsCas12f1-N70Q amino acid sequence preferably comprises the sequence shown below: SEQ ID No.3

[0337] The variant AsCas12f1-K103R amino acid sequence preferably comprises the sequence shown below: SEQ ID No.4

[0338] The variant AsCas12f1 A104R amino acid sequence preferably comprises the sequence shown below: SEQ ID No.5

[0339] The variant AsCas12f1-K107R amino acid sequence preferably comprises the sequence shown below: SEQ ID No.6

[0340] The variant AsCas12f1-A E108R amino acid sequence preferably comprises the sequence shown below: SEQ ID No.7

[0341] The variant AsCas12f1-S115R amino acid sequence preferably comprises the sequence shown below: SEQ ID No.8

[0342] The variant AsCas12f1-S118A amino acid sequence preferably comprises the sequence shown below: SEQ ID No.9

[0343] The variant AsCas12f1-D364R amino acid sequence preferably comprises the sequence shown below: SEQ ID No.10

[0344] The underlined 20bp sequence in the plasmid sequence can be reasonably designed and replaced according to the target site editing needs. The target site should be located on the target gene, and the 5' upstream of the target site should contain a preferred PAM sequence, and its sequence characteristics should be 5'-TTR, where R represents A or G. The target sequence in the plasmid can be replaced by conventional technical methods in the art, such as Goldengate assembly.

[0345] In this embodiment, the target sequence is as follows:

[0346] SEQ ID No.46:TTTG_1:5'-CTCTCAAGACCCACAATCCA-3'

[0347] SEQ ID No.47:TTTG_2:5'-CTGTCGACACCCAGAATCAT-3'

[0348] SEQ ID No.48:CTTG_1:5'-TCCTGCTTGCTTACCTCGCT-3'

[0349] SEQ ID No.49:CTTG_2:5'-CCTCTTTCCTAGCACTGCCC-3'

[0350] SEQ ID No.50:ATTG_1:5'-AAGAAGGGATGTGGTGCATT-3'

[0351] SEQ ID No.51:ATTG_2:5'-CTGTGATTTCCCCACAAAAG-3'

[0352] SEQ ID No.52:TTTA_1:5'-CCTGTGAGATAAGGCCAGTA-3'

[0353] SEQ ID No.53:TTTA_2:5'-TCTGTCCCCTCCACCCCACA-3'

[0354] In this example, human embryonic kidney cells HEK293T were used as experimental cells.

[0355] The activated HEK293T cells were cultured in DMEM medium containing 10% FBS, and the cells were subcultured into 24-well plates when the cell density reached about 90%. The number of cells per well was about 1.0×10 5 After 16-18 hours, 1000ng of gene editing plasmids expressing wild-type AsCas12f1 nuclease and sgRNA_T1 with different target sequences were transfected into the cells with 1.5μL of lipofectamine3000. After 48 hours of culture, the adherent cells were digested and genomic DNA was extracted. The target gene fragment of the targeted sequence was amplified by PCR, and the PCR product after gel recovery was annealed with NEBuffer2 (NEB). T7 endonuclease 1 was then added to the reaction system, and 6×Gel Loading Dye was added after enzyme digestion at 37℃ for 15min to terminate the reaction. The reaction products were separated by 6% TBE-PAGE and stained with 4S Red dye for imaging.

[0356] Figure 1 This is a comparison of the gene editing results of mammalian cells by wild-type AsCas12f1 and mutant AsCas12f1 nucleases. As shown in the figure, the mutant AsCas12f1 nuclease single mutant can significantly improve the gene editing efficiency of eight target sites, TTTG_1, TTTG_2, CTTG_1, CTTG_2, ATTG_1, ATTG_2, TTTA_1, and TTTA_2, with the highest improvement ratio being 3 times.

[0357] Example 2 Combination mutation of mutant AsCas12f1 nuclease for mammalian cell gene editing

[0358] Using the pCMV-AsCas12f1-T1 plasmid constructed in Example 1 as a template, polymerase chain reaction was performed using point mutation primers for different amino acid residues to obtain 13 AsCas12f1 nuclease combination variant plasmids with multiple amino acid residue point mutations, which can be used for mammalian cell gene editing.

[0359] The primers used to construct the plasmid of the AsCas12f1 nuclease combination variant are shown in Example 1.

[0360] The PCR products were digested with Dpn1 and then recovered from the solution. After being assembled by the Golden gate assembly technology, they were transformed into Escherichia coli DH5α competent cells, coated on LBA plates with ampicillin, single clones were picked for expansion culture, plasmids were extracted, and sequencing and identification were performed to obtain plasmids encoding AsCas12f1-N70Q+A104R, AsCas12f1-K103R+A104R, AsCas12f1-A104R+K107R, AsCas12f1-A104R+E108R, AsCas12f1-A104R+S115R, AsCas12f1-A104R+S118A, AsCas12f1-A104R+D364R, AsCas12f1-N pCMV-AsCas12f1-T1 mammalian cell gene editing plasmids containing 70Q+A104R+D364R, AsCas12f1-N70Q+A104R+S115R+D364R, AsCas12f1-N70Q+A104R+S118A+D364R, AsCas12f1-N70Q+K103R+A104R+S118A+D364R, AsCas12f1-N70Q+A104R+K107R+S118A+D364R, and AsCas12f1-N70Q+K103R+A104R+K107R+S118A+D364R.

[0361] The variant AsCas12f1-N70Q+A104R amino acid sequence preferably comprises the sequence shown below: SEQ ID No.11

[0362] The variant AsCas12f1-K103R+A104R amino acid sequence preferably comprises the following sequence: SEQ ID No. 12

[0363] The variant AsCas12f1-A104R+K107R amino acid sequence preferably comprises the following sequence: SEQ ID No. 13

[0364] The variant AsCas12f1-A104R+E108R amino acid sequence preferably comprises the following sequence: SEQ ID No. 14

[0365] The variant AsCas12f1 A104R+S115R amino acid sequence preferably comprises the following sequence: SEQ ID No. 15

[0366] The variant AsCas12f1-A104R+S118A amino acid sequence preferably comprises the following sequence: SEQ ID No. 16

[0367] The variant AsCas12f1-A104R+D364R amino acid sequence preferably comprises the sequence shown below: SEQ ID No. 17

[0368] The variant AsCas12f1-N70Q+A104R+D364R amino acid sequence preferably comprises the following sequence: SEQ ID No. 18

[0369] The variant AsCas12f1-N70Q+A104R+S115R+D364R amino acid sequence preferably comprises the following sequence: SEQ ID No.19

[0370] The variant AsCas12f1-N70Q+A104R+S118A+D364R amino acid sequence preferably comprises the following sequence: SEQ ID No.20

[0371] The variant AsCas12f1-N70Q+K103R+A104R+S118A+D364R amino acid sequence preferably comprises the following sequence: SEQ ID No.21

[0372] The variant AsCas12f1-N70Q+A104R+K107R+S118A+D364R amino acid sequence preferably comprises the following sequence: SEQ ID No.22

[0373] The variant AsCas12f1-N70Q+K103R+A104R+K107R+S118A+D364R amino acid sequence preferably comprises the following sequence: SEQ ID No.23

[0374] Referring to the target sequence replacement method in Example 1, the target sequences TTTG_1, TTTG_2, CTTG_1, CTTG_2, ATTG_1, ATTG_2, TTTA_1, and TTTA_2 were inserted into each variant plasmid, respectively.

[0375] Referring to Example 1, gene editing tests were performed using human embryonic kidney cells HEK293T as experimental cells.

[0376] Figure 2 The results of mammalian cell gene editing of the engineered optimized AsCas12f1 nuclease combination mutants. As shown in the figure, the 13 combination variants can significantly improve the gene editing efficiency of eight target sites, TTTG_1, TTTG_2, CTTG_1, CTTG_2, ATTG_1, ATTG_2, TTTA_1, and TTTA_2, under four PAMs: 5'-TTTG, 5'-CTTG, 5'-ATTG, and 5'-TTTA. Among them, AsCas12f1-N70Q+K103R+A104R+S118A+D364 (v5.1) has the best effect, with the highest improvement ratio of 13 times.

[0377] Example 3 Application of mutant AsCas12f1 gene editing system in gene editing in mammalian cells

[0378] The engineered optimized AsCas12f1 nuclease variant described in this example is AsCas12f1-v5.1.

[0379] The engineered optimized CRISPR / AsCas12f1 gene editing system described in this embodiment is a combination of AsCas12f1-v5.1 and sgRNA_T1.

[0380] Referring to the target sequence replacement method in Example 1, the target sequences ATTA_1-10, CTTA_1-19, GTTA_1-11, TTTA_1-16, ATTG_1-15, CTTG_1-24, GTTG_1-11, and TTTG_1-32 were respectively inserted into the pCMV-AsCas12f1-T1-N70Q+K103R+A104R+S118A+D364R (v5.1) plasmid as follows:

[0381] SEQ ID No.54:ATTA_1:5'-AACAGCCCTGAAGTGTTTCT-3'

[0382] SEQ ID No.55:ATTA_2:5'-ACCGGCCCTGGGAATATAAG-3'

[0383] SEQ ID No.56:ATTA_3:5′-ACAGTTTGCTTATAAAACTT-3′SEQ ID No.57:ATTA_4:5′-ATGAGGCTACTAGATCATTT-3′SEQ ID No.58:ATTA_5:5′-TGCCCACCTGTGCCATTCTG-3′SEQ IDNo.59:ATTA_6:5'-ATAACTAAGGTTTTGTGGCA-3'SEQ ID No.60:ATTA_7:5'-ATACAAACTATCATCCCTGC-3'SEQ ID No.61:ATTA_8:5'-CGTATTTTCACAAGTTTTTT-3'SEQ IDNo.62:ATTA_9:5'-GGTATAAATACCAGCAGCCA-3'SEQ ID No.63:ATTA_10:5′-TGGCGAACCTTGGCTGCTGG-3′SEQ ID No.64:CTTA_1:5′-GTGCTCCCTGGGGGCAGCTC-3′SEQ IDNo.65:CTTA_2:5′-CCTCGCTTAGTGCTCCCTGG-3′SEQ ID No.66:CTTA_3:5′-TCAAGTGGAAAGTTTCCAGT-3′SEQ ID No.67:CTTA_4:5′-GGGAAGCGGGACCCTGCTCT-3′SEQ IDNo.68:CTTA_5:5′-AAGAGTCCCCAGTGCTATCT-3′SEQ ID No.69:CTTA_6:5′-TTCTGATTTTGTTTTTCCAA-3′SEQ ID No.70:CTTA_7:5′-CACTTCCCAAGAGGAGAAGC-3′SEQ IDNo.71:CTTA_8:5′-CGATGGAGCCAGAGAGGATC-3′SEQ ID No.72:CTTA_9:5′-GAGGTTCTGGCAAGGAGAGA-3′SEQ ID No.73:CTTA_10:5′-TCTGGTGACACACCCCCATT-3′SEQ IDNo.74:CTTA_11:5′-GGCCTCCTCCTTCCTAGTCT-3′SEQ ID No.75:CTTA_12:5'-TATTCCCAGGGCCGGTTAAT-3'SEQ ID No.76:CTTA_13:5'-CCTCTCTAGTCTGTGCTAGC-3'SEQ IDNo.<h2 style=";text-align:left;direction:ltr">77:CTTA_14:5'-GGATGGCCTTCTCCGACGGA-3'SEQ ID No.78:CTTA_15:5'-TCAGGCCATGATTCTGGGTG-3'SEQ ID No.79:CTTA_16:5'-AGTCCTTCTTGACTGACCTT-3'SEQ ID No.80:CTTA_17:5'-AGCTCTCAAATGACATGATG-3'SEQ ID No.81:CTTA_18:5'-TCACTGGACTTCTCTTCAAA-3'SEQ ID No.82:CTTA_19:5'-AAAAACTTGTGAAAATACGT-3'SEQ ID No.83:GTTA_1:5'-TGCCTCAGATTCACTTTTAT-3'SEQ ID No.84:GTTA_2:5'-GACTGGAAACTTTCCACTTG-3'SEQ ID No.85:GTTA_3:5'-GAACTCAGGACCAACTTATT-3'SEQ ID No.86:GTTA_4:5'-GGCAGATTCCTTATCTGGTG-3'SEQ ID No.87:GTTA_5:5'-GACCCAATATCAGGAGACTA-3'SEQ IDNo.89:GTTA_7:5'-ATTTCATAGATGCCCCAGAG-3'SEQ ID No.90:GTTA_8:5'-ATTTACAGCTACAGCCCTAT-3'.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0384] SEQ ID No.91:GTTA_9:5'-CCAGAATAGGGCTGTAGC-3'SEQ ID No.92:GTTA_10:5'-GTAAAATATCTACCACAAGT-3'SEQ ID No.93:GTTA_11:5'-GAGTTTCCTTTAGACTCCTT-3'SEQID No.94:TTTA_1:5'-CCTGTGAGATAAGGCCAGTA-3'SEQ ID No.95:TTTA_2:5'-TCTGTCCCCTCCACCCCACA-3'SEQ ID No.96:TTTA_3:5'-CCAGGTTGAGTCGCTTTGGT-3'SEQ IDNo.97:TTTA_4:5'-CTAGGGGTCCTCTGTGTGAA-3'SEQ ID No.98:TTTA_5:5'-TGCATCTCTGGGTCCCCGGC-3'SEQ ID No.99:TTTA_6:5'-TCACCTTTCCTTGCCTCTTT-3'

[0385] SEQ ID No.100:TTTA_7:5'-CCTGCAATTGGGGCATTTGC-3'SEQ ID No.101:TTTA_8:5'-AGGAAAGAAGGATGGAGAAA-3'SEQ ID No.102:TTTA_9:5'-GCCACCTCTCCATCCTCTTG-3'SEQ ID No.103:TTTA_10:5'-CAGCTCTGACAAGTTTTATA-3'SEQ ID No.104:TTTA_11:5'-AATTTACCTGTGTGGCTTGT-3'SEQ ID No.105:TTTA_12:5'-CCTGTGTGGCTTGTATTGTA-3'SEQ ID No.106:TTTA_13:5'-ACCGCATTCTTCCTTGCTT-3'SEQ ID No.107:TTTA_14:5'-AAAGGCTGGCCCTTTGTAAA-3'SEQ ID No.108:TTTA_15:5'-CAAAGGGCAGCCTTTTAAA-3'SEQ IDNo.109:TTTA_16:5'-GACTCCTTGGGTCCTTTGAC-3'SEQ ID No.110:ATTG_1:5'-AAGAAGGGATGTGGTGCATT-3'SEQ ID No.111:ATTG_2:5'-CTGTGATTTCCCCACAAAAG-3'SEQ IDNo.112:ATTG_3:5'-TGGGTCTTGAGAGCAAAAAC-3'SEQ ID No.113:ATTG_4:5'-TCTTTGAGGCATCACTGCCC-3'SEQ ID No.114:ATTG_5:5'-TTCCTCCGTGCGTCAGTTTT-3'SEQ IDNo.115:ATTG_6:5'-GGGACTAGAAAGGTGAAGAG-3'SEQ ID No.116:ATTG_7:5'-GGTCTAACCCCCACCTCCTG-3'SEQ ID No.117:ATTG_8:5'-GTGACAGAAAAGCCCCATCC-3'SEQ IDNo.118:ATTG_9:5'-TGTTCCCTGAAGCGGCCATT-3'

[0386] SEQ ID No.119:ATTG_10:5'-CAGGCTTATCACTGGACTTC-3'SEQ ID No.120:ATTG_11:5'-TATTTCTACTGGGCAGTGCT-3'SEQ ID No.121:ATTG_12:5'-CTCTAGATCAGCACTGCCCA-3'SEQ ID No.122:ATTG_13:5'-TTCTTTTAACCGCATTCTTT-3'

[0387] SEQ ID No.123:ATTG_14:5'-ATCATTTGCTTGTGGTTGGG-3'SEQ ID No.124:ATTG_15:5'-ACATGTTAAAAACACAACAT-3'SEQ ID No.125:CTTG_1:5'-TCCTGCTTGCTTACCTCGCT-3'

[0388] SEQ ID No.126:CTTG_2:5'-CCTCTTTCCTAGCACTGCCC-3'

[0389] SEQ ID No.127:CTTG_3:5'-AGAGCAAAAACCTGTTTTAT-3'SEQ ID No.128:CTTG_4:5'-CCTCTTCCGGCCTGGATTGT-3'

[0390] SEQ ID No.129:CTTG_5:5'-GGGAAATGATTCTTTTGTGG-3'SEQ ID No.130:CTTG_6:5'-CTTACCTCGCTTAGTGCTCC-3'

[0391] SEQ ID No.131:CTTG_7:5'-GTCTCCTCCACCGCTTCTTG-3'

[0392] SEQ ID No.132:CTTG_8:5’-ATAAGAGGTCCCAAGACTTA-3’SEQ ID No.133:CTTG_9:5’-GGACCTCTTATCAAGTGGAA-3’SEQ ID No.134:CTTG_10:5’-ACTGTTTTACCTGCAATTGG-3’SEQID No.135:CTTG_11:5’-GCAAGCCCAGGAGAGGCGCT-3’SEQ ID No.136:CTTG_12:5’-CCAAGGACTCAAACCCAGAA-3’SEQ ID No.137:CTTG_13:5’-GGAAGTGTAAGGAAGCTGCA-3’SEQ IDNo.138:CTTG_14:5’-GCAGGGGGTGGGAGGGAAGG-3’SEQ ID No.139:CTTG_15:5’-CCAGAACCTCTAAGGTTTGC-3’SEQ ID No.140:CTTG_16:5’-CTTTCTTTGCCTGGACACCC-3’SEQ IDNo.141:CTTG_17:5’-TAGGCCTGCATCATCACCGT-3’SEQ ID No.142:CTTG_18:5’-CGTCCCGCCTCCCCTTCTTG-3’SEQ ID No.143:CTTG_19:5’-GCCTCTCTGGGGCATCTATG-3’SEQ IDNo.144:CTTG_20:5’-AAGTTGAAAATGTTTTTGCT-3’SEQ ID No.145:CTTG_21:5’-ACTGACCTTGAAGTTGAAAA-3’SEQ ID No.146:CTTG_22:5’-ACAACATTTACAGCTCTGAC-3’SEQ IDNo.147:CTTG_23:5’-CTTTCTGGTCATTTGCAAGA-3’SEQ ID No.148:CTTG_24:5’-TGGTTGGGAACAAGTGAAAA-3’SEQ ID No.149:GTTG_1:5’-AGTCGCTTTGGTGAGGTGTG-3’SEQ IDNo.150:GTTG_2:5’-GGCAGTGCTAGGAAAGAGGC-3’SEQ ID No.151:GTTG_3:5’-GTCCTGAGTTCTAACTTTGG-3’SEQ ID No.152:GTTG_4:5'-GAGGAAAGACTAGCTGAG-3'SEQ IDNo.153:GTTG_5:5'-TCCAGAAAAACGGTGATGAT-3'SEQ ID No.154:GTTG_6:5'-TTAATTTCATAGATGCCCCA-3'SEQ ID No.155:GTTG_7:5'-TCAAGTTTTGAGAGAAGTC-3'SEQ IDNo.156:GTTG_8:5'-GTTAAGAAATTCTACCAC-3'SEQ ID No.157:GTTG_9:5'-TAGTTAGAGTTTCCTTTAGA-3'SEQ ID No.158:GTTG_10:5'-GAACTGAGTCCCAAGATGTG-3'SEQ IDNo.159:GTTG_11:5'-GACGAAAAGTTTCAGTGCGA-3'SEQ ID No.160:TTTG_1:5'-CTGTGACACCAGAATTCAT-3'SEQ No.161:TTTG_2:5'-CTCTCAAGACCCACAATCCA-3'SEQ IDNo.162:TTTG_3:5'-AGTATACGCTTCCACAGAAA-3'SEQ ID No.163:TTTG_4:5'-CTGTGAGCTCTAGTCCCCAC-3'SEQ ID No.164:TTTG_5:5'-AGGCATCACTGCCCCCTGAT-3'SEQ IDNo.165:TTTG_6:5'-GTGAGGTGTGAACATGGA-3'SEQ ID No.166:TTTG_7:5'-GCTGGGGAGGAGCTGQGTG IDNo.165:TTTG_6:5'-GTGAGGTGTGAACATGGA-3' No.167:TTTG_8:5'-CCATCAGGGGCAGTGATGC-3'SEQ IDNo.168:TTTG_9:5'-CTTACGATGGAGCCAGAG-3'SEQ ID No.169:TTTG_10:5'-CTGCCTCCAGGGATCCTSEQGTG-3' No.170:TTTG_11:5'-GGCAGCTCCCCTACCCCCCT-3'SEQ IDNo.171:TTTG_12:5'-CCTGGACACCCCGTTCTCCT-3'SEQ ID No.172:TTTG_13:5'-TTGTTAATTTCAGATSEQGCC IDNo.3'173:TTTG_14:5’-CATTGTGTTCCCTGAAGCGG-3’SEQ IDNo.174:TTTG_15:5’-AGAGCTTAAGTCCTTCTTGA-3’SEQ ID No.175:TTTG_16:5’-ACCACACAAACAGTCTGAAC-3’SEQ ID No.176:TTTG_17:5’-TGTGGTCAAATTTCATTTCA-3’.

[0393] SEQ ID No.177:TTTG_18:5’-AAACTTGTGGTAGATATTTT-3’

[0394] SEQ ID No.178:TTTG_19:5’-GTGAGGAAGTCCTTCATCAG-3’

[0395] SEQ ID No.179:TTTG_20:5’-CAAGAAAAATTTTAAAAGGC-3’

[0396] SEQ ID No.180:TTTG_21:5’-TAAAGGTTTGAGAGGCCCTA-3’

[0397] SEQ ID No.181:TTTG_22:5’-AGAGGCCCTAGAATTTCGTT-3’

[0398] SEQ ID No.182:TTTG_23:5’-CTTGTGGTTGGGAACAAGTG-3’

[0399] SEQ ID No.183:TTTG_24:5’-TGAATGAAGAGTCAACATTT-3’

[0400] SEQ ID No.184:TTTG_25:5’-TACCTCCCCACTTCGCCCTG-3’

[0401] SEQ ID No.185:TTTG_26:5’-ACGATGAGACAGACCCATTA-3’

[0402] SEQ ID No.186:TTTG_27:5’-TGGCCACATGGAGTGACCTG-3’

[0403] SEQ ID No.187:TTTG_28:5’-TGTGGCCACAAGGCTCAGTT-3’

[0404] SEQ ID No.188:TTTG_29:5'-CCCAGAGCATCCCGTGGAAC-3'

[0405] SEQ ID No.189:TTTG_30:5'-CCTTGTTCCGATTCAGTCAT-3'

[0406] SEQ ID No.190:TTTG_31:5'-CCTTGTCAAGGCTATTGGTC-3'

[0407] SEQ ID No.191:TTTG_32:5'-GGATAAGCACAGTTTTAAAT-3'

[0408] Referring to Example 1, gene editing tests were performed using human embryonic kidney cells HEK293T as experimental cells.

[0409] Figure 3 This is a comparison of the gene editing effects of the wild-type CRISPR / AsCas12f1 system and the mutant CRISPR / AsCas12f1 system based on the nuclease of sgRNA_T1 in mammalian cells. As shown in the figure, the CRISPR / AsCas12f1 gene editing system after engineering optimization can significantly improve the gene editing efficiency at 138 target sites with PAMs of 5'-CTTA, 5'-ATTA, 5'-GTTA, 5'-TTTA, 5'-ATTG, 5'-CTTG, 5'-GTTG, and 5'-TTTG. Among them, the highest improvement ratio is 100 times.

[0410] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. A mutant AsCas12f1 nuclease, characterized in that The mutant AsCas12f1 nuclease comprises one or more point mutations in amino acids 8, 69, 70, 103, 104, 107, 108, 115, 118, and 364 relative to the wild-type AsCas12f1 nuclease; the nucleotide sequence encoding the wild-type AsCas12f1 nuclease is shown in SEQ ID No.

24.

2. The mutant AsCas12f1 nuclease according to claim 1, characterized in that The point mutation has one or more of the following characteristics: 1) The glutamic acid E at position 8 in the wild-type AsCas12f1 nuclease was mutated to arginine R; 2) The threonine T at position 69 in the wild-type AsCas12f1 nuclease was mutated to serine S; 3) Asparagine N at position 70 in the wild-type AsCas12f1 nuclease was mutated to glutamine Q; 4) Lysine K at position 103 in the wild-type AsCas12f1 nuclease was mutated to arginine R; 5) Alanine A at position 104 in the wild-type AsCas12f1 nuclease was mutated to arginine R; 6) Lysine K at position 107 in the wild-type AsCas12f1 nuclease was mutated to arginine R; 7) The glutamic acid E at position 108 in the wild-type AsCas12f1 nuclease was mutated to arginine R; 8) The serine S at position 115 in the wild-type AsCas12f1 nuclease was mutated to arginine R; 9) The serine S at position 118 in the wild-type AsCas12f1 nuclease was mutated to alanine A; 10) Aspartic acid D at position 364 in the wild-type AsCas12f1 nuclease was mutated to arginine R.

3. The mutant AsCas12f1 nuclease according to claim 2, characterized in that The point mutation has one or more of the following characteristics: 1) The 70th and 104th positions in the wild-type AsCas12f1 nuclease were mutated simultaneously; 2) The 103rd and 104th positions in the wild-type AsCas12f1 nuclease were mutated simultaneously; 3) The 104th and 107th positions in the wild-type AsCas12f1 nuclease were mutated simultaneously; 4) The 104th and 108th positions in the wild-type AsCas12f1 nuclease were mutated simultaneously; 5) The 104th and 115th positions in the wild-type AsCas12f1 nuclease were mutated simultaneously: 6) The wild-type AsCas12f1 nuclease contains the point mutations at positions 104 and 118: 7) The 104th and 364th positions in the wild-type AsCas12f1 nuclease were mutated simultaneously: 8) The 70th, 104th and 364th positions in the wild-type AsCas12f1 nuclease were mutated simultaneously; 9) Mutations at positions 70, 104, 115 and 364 in the wild-type AsCas12f1 nuclease: 10) mutations at positions 70, 104, 118, and 364 in the wild-type AsCas12f1 nuclease; 11) The 70th, 103rd, 104th, 118th and 364th positions in the wild-type AsCas12f1 nuclease were mutated simultaneously; 12) The 70th, 104th, 107th, 118th and 364th positions in the wild-type AsCas12f1 nuclease were mutated simultaneously: 13) Positions 70, 103, 104, 107, 118 and 364 in the wild-type AsCas12f1 nuclease were mutated simultaneously.

4. The mutant AsCas12f1 nuclease according to claim 1, characterized in that The mutant AsCas12f1 nuclease comprises an amino acid sequence as shown in any one of SEQ ID No.1-23.

5. An isolated polynucleotide, characterized in that The polynucleotide comprises a nucleotide sequence encoding the mutant AsCas12f1 nuclease described in any one of claims 1-3.

6. The polynucleotide according to claim 5, characterized in that The polynucleotide comprises a nucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID No. 1-23.

7. A nucleic acid construct, characterized in that The nucleic acid construct comprises the polynucleotide according to any one of claims 5-6.

8. The nucleic acid construct according to claim 7, characterized in that The nucleic acid construct further comprises a nucleotide sequence encoding a gRNA or a regulatory element; preferably, the regulatory element is selected from a promoter, an enhancer, a nuclear localization signal or a poly A structure.

9. A gene editing system, characterized in that: The gene editing system comprises the mutant AsCas12f1 nuclease and gRNA or a polynucleotide encoding gRNA as described in any one of claims 1-4; or, comprises the polynucleotide and gRNA or a polynucleotide encoding gRNA as described in any one of claims 5-6; or, comprises the nucleic acid construct and gRNA or a polynucleotide encoding gRNA as described in claim 7; or, comprises the nucleic acid construct as described in claim 8.

10. The gene editing system according to claim 8, characterized in that: The gene editing system recognizes the PAM sequence on the targeting sequence; preferably the PAM sequence is 5'-TTR, where R represents A or G.

11. A composition, characterized in that The composition comprises the mutant AsCas12f1 nuclease described in any one of claims 1-4, the polynucleotide described in any one of claims 5-6, the nucleic acid construct described in any one of claims 7-8 or the gene editing system described in any one of claims 9-10, and a pharmaceutically acceptable carrier or excipient.

12. An in vitro gene editing method, characterized in that: The method contacts the target gene with the gene editing system described in any one of claims 8-9 to edit the target gene.

13. Use of the mutant AsCas12f1 nuclease described in any one of claims 1-4, the polynucleotide belonging to any one of claims 5-6, the nucleic acid construct described in any one of claims 7-8, the gene editing system described in any one of claims 9-10, or the composition described in claim 11 for gene editing of a target gene in an in vitro cell or a cell-free environment.

14. A genetically modified cell, characterized in that The cell is obtained by genetically modifying an in vitro cell by gene editing using the mutant AsCas12f1 nuclease described in any one of claims 1-4, the polynucleotide described in any one of claims 5-6, the nucleic acid construct described in any one of claims 7-8, the gene editing system described in any one of claims 9-10, or the composition described in claim 11, or the gene editing method described in claim 12.

15. Use of the mutant AsCas12f1 nuclease according to any one of claims 1 to 3 in the preparation of any of the following systems: 1) Single-base editing system based on mutant AsCas12f1 nuclease and base deaminase; 2) Prime editing system based on mutant AsCas12f1 nuclease and reverse transcriptase; 3) Transcriptional activation system based on mutant AsCas12f1 nuclease and transcriptional activator; 4) Epigenetic modification system based on mutant AsCas12f1 nuclease and epigenetic modification system; 5) Transcription inhibition system based on inactivated mutant AsCas12f1 nuclease.

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