Cas9 nuclease and preparation method and use thereof

Abstract

The present invention relates to the field of biotechnology, and in particular to a Cas9 nuclease and a preparation method and use thereof. The Cas9 nuclease provided by the present invention comprises a polypeptide formed by one or more amino acid mutations at position 915 and / or position 976 of a wild-type Cas9 nuclease; the amino acid sequence of the wild-type Cas9 nuclease is shown in SEQ ID No. 2. Compared with the wild-type Cas9 nuclease, the Cas9 nuclease (DE Cas9) provided by the present invention can produce a larger proportion of DNA deletions and a smaller proportion of DNA insertions by cutting a DNA double strand using DE Cas9, which can change the DNA editing and repair results of the Cas9 nuclease.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Cas9 nuclease and a preparation method and use thereof. Background Art

[0002] Since the completion of the Human Genome Project and the Encyclopedia of DNA Elements, scientists have analyzed and identified a large number of genes and DNA regulatory elements in the genome [1,2]. DNA regulatory elements that play an important role in gene expression regulation include promoters, enhancers, silencers, and insulators. However, the functions of most regulatory elements have not been experimentally verified and elucidated [2-8]. To explore the functions of genes and DNA regulatory elements, genetic DNA fragment editing can be used for research.

[0003] Early gene editing and gene function modification were achieved through gene transposition and transgenesis [9-14]. With the development of sequencing technology, reverse genetics was applied to specific mutations in the genome [15,16]. In particular, gene-targeted mice that rely on homologous recombination were rapidly applied to scientific research [15,17,18]. In addition, the inversion and duplication of DNA fragments in mice and zebrafish were used to study specific genomic structural changes [19-24].

[0004] In recent years, the type II clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9) CRISPR / Cas9, which originated from bacteria and archaea, is an emerging genome editing technology [25-27]. Due to its simple design and convenient operation, it has been rapidly applied to eukaryotic genome editing. In CRISPR / Cas9 genome editing technology, sgRNA guides the Cas9 protein to its target site, and finally generates double-strand DNA breaks (DSB) at the target site. The breaks are repaired through endogenous repair pathways, including nonhomologous end-joining (NHEJ) and microhomology-mediated end joining (MMEJ), as well as homology-directed repair (HDR) using repair templates, which has more accurate repair results, and may ultimately produce different repair results

[28] . In addition to the traditional CRISPR / Cas9-induced double-stranded DNA cleavage knockout, new technologies are emerging in an endless stream. For example, base editing provides a strategy to generate site-specific and precise point mutations without DSBs. Due to its accuracy and efficiency, it shows great potential in the treatment of genetic diseases caused by certain single-base mutations. However, gene mutations in many human genetic diseases are not limited to single-base mutations. Frameshift mutations are also a very important type of mutation. At the same time, base editors cannot achieve the purpose of efficiently restoring normal sequences. In recent years, in vitro cleavage experiments of Cas9 nucleases have revealed that the RuvC nuclease domain cuts the non-complementary strand of the targeted DNA with diversity, which can break at -3, -4 or further positions upstream of PAM. At the same time, the HNH nuclease domain cuts the complementary strand at a fixed position of 3bp upstream of PAM, thereby generating a 5' overhang. This cleavage mode may produce a loss of DNA fragments through DNA repair in cells. Using this cleavage feature, diseases caused by certain specific base insertions can be more efficiently edited and restored to a normal genotype. This method does not require the addition of additional templates and has great application prospects in disease treatment. How to increase the proportion of DNA fragment deletions in editing results is one of the important issues.

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[28] Summary of the invention

[0034] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a Cas9 nuclease and a preparation method and use thereof, so as to solve the problems in the prior art.

[0035] To achieve the above-mentioned and other related purposes, the present invention provides a Cas9 nuclease, wherein the Cas9 nuclease comprises a polypeptide formed by one or more amino acid mutations at position 915 and / or position 976 of a wild-type Cas9 nuclease; the amino acid sequence of the wild-type Cas9 nuclease is shown in SEQ ID No. 2

[0036] Preferably, the Cas9 nuclease comprises a polypeptide having an amino acid sequence such as SEQ ID No.1.

[0037] The present invention also provides an isolated polynucleotide encoding the aforementioned Cas9 nuclease.

[0038] The present invention also provides an expression vector, which comprises the aforementioned polynucleotide and a plasmid backbone.

[0039] The present invention also provides a gene editing system, which comprises the aforementioned Cas9 nuclease, and gRNA or a nucleic acid fragment encoding gRNA; or, comprises the aforementioned polynucleotide, and gRNA or a polynucleotide encoding gRNA; or, comprises the aforementioned expression vector, and gRNA or a polynucleotide encoding gRNA.

[0040] The present invention also provides a cell, which contains the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned gene editing system.

[0041] The present invention also provides a method for preparing the aforementioned Cas9 nuclease, which comprises: introducing the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned gene editing system into an engineered cell, inducing the expression of the exogenous gene, and separating and purifying to obtain the Cas9 nuclease.

[0042] The present invention also provides the use of the aforementioned Cas9 nuclease, the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned gene editing system for performing gene editing on a target gene in an in vitro cell or a cell-free environment.

[0043] The present invention also provides an in vitro gene editing method, which comprises: contacting a target gene with the aforementioned gene editing system to achieve editing of the target gene.

[0044] As described above, a Cas9 nuclease and a preparation method and use thereof of the present invention have the following beneficial effects: Compared with the wild-type Cas9 nuclease, the Cas9 nuclease (DE Cas9) provided by the present invention can produce a larger proportion of DNA deletions and a smaller proportion of DNA insertions by cutting the DNA double strands using DE Cas9, which can change the DNA editing and repair results of the Cas9 nuclease. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It shows the proportion of deletions generated by detecting each Cas9 nuclease when cutting the genomic DNA fragment under the mediation of ATN1 sgRNA4 in the present invention.

[0046] Figure 2 It shows the proportion of deletions produced by detecting each Cas9 nuclease when cutting the genomic DNA fragment under the mediation of ATN1 sgRNA5 in the present invention.

[0047] Figure 3 It shows the percentage of deletions generated when WT-Cas9, G915F, R976A, and DE Cas9 nucleases cut genomic DNA fragments under the mediation of sgRNA4 by detecting the connection of DNA fragment deletion adapters in the present invention.

[0048] Figure 4 It shows the percentage of deletions generated when WT-Cas9, G915F, R976A, and DE Cas9 nucleases cut genomic DNA fragments under the mediation of sgRNA5 by detecting the connection of DNA fragment deletion adapters in the present invention.

[0049] Figure 5 It shows the proportion of various indels generated when WT-Cas9, G915F, R976A, and DE Cas9 nucleases cut genomic DNA fragments under the mediation of sgRNA4 by detecting the connection of DNA fragment deletion adapters in the present invention.

[0050] Figure 6It shows the proportion of various indels generated when WT-Cas9, G915F, R976A, and DE Cas9 nucleases cut genomic DNA fragments under the mediation of sgRNA5 by detecting the connection of DNA fragment deletion adapters in the present invention.

[0051] Figure 7 It shows the percentage of insertions generated when WT-Cas9, G915F, R976A, and DE Cas9 nucleases cut genomic DNA fragments under the mediation of sgRNA4 by detecting the connection of DNA fragment deletion adapters in the present invention.

[0052] Figure 8 It shows the percentage of insertions generated when WT-Cas9, G915F, R976A, and DE Cas9 nucleases cut genomic DNA fragments under the mediation of sgRNA5 by detecting the connection of DNA fragment deletion adapters in the present invention. DETAILED DESCRIPTION

[0053] The present invention provides a Cas9 nuclease, which comprises a polypeptide formed by one or more amino acid mutations of a wild-type Cas9 nuclease at position 915 and / or position 976. The amino acid sequence of the wild-type Cas9 nuclease is shown in SEQ ID No. 2.

[0054] In some specific embodiments, the nucleic acid molecule encoding the wild-type Cas9 nuclease comprises a nucleic acid fragment with a nucleotide sequence as shown in SEQ ID No.4.

[0055] In some embodiments, the amino acid mutation comprises one or more of the following features:

[0056] I) mutation of glycine to glutamic acid, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, lysine, arginine or histidine;

[0057] II) Arginine mutation to glycine, glutamic acid, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, asparagine, glutamine, threonine, aspartic acid, lysine, phenylalanine or histidine.

[0058] Furthermore, the amino acid mutation is from glycine to phenylalanine; and / or, arginine is mutated to alanine.

[0059] Furthermore, the amino acid mutation is that the wild-type Cas9 nuclease mutates from glycine to phenylalanine at position 915; and / or the wild-type Cas9 nuclease mutates from arginine to alanine at position 976.

[0060] In some specific embodiments, the Cas9 nuclease comprises a polypeptide having an amino acid sequence as shown in SEQ ID No.1.

[0061] The present invention also provides an isolated polynucleotide encoding the aforementioned Cas9 nuclease.

[0062] In some specific embodiments, the polynucleotide comprises a nucleic acid fragment whose nucleotide sequence is shown as SEQ ID No.3.

[0063] The present invention also provides an expression vector, which comprises the aforementioned polynucleotide and a plasmid backbone.

[0064] In some embodiments, the plasmid backbone comprises an operable element. Specifically, the operable element comprises one or more of a promoter, an enhancer, a terminator, a silencer, a nuclear localization signal, a poly A structure, a reporter gene or a transposon.

[0065] In some specific embodiments, the plasmid backbone is selected from pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-Tag One or more of FP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP / VSV-G, pENTR / U6, pLenti6 / BLOCK-iT-DEST, pcDNA1.2 / V5-GW / lacZ, pLenti6.2 / N-Lumio / V5-DEST, pGCSIL-GFP, pAAV-CAG, pAAV-TRE, pAAV-EF1a, pAAV-GFAP, pAAV-Lgr5, pAAV-Sox2, pAAV-Syn or pAAV-CMV.

[0066] The present invention also provides a gene editing system, which comprises the aforementioned Cas9 nuclease and a guide RNA (gRNA) or a nucleic acid fragment encoding a guide RNA (gRNA); or, comprises the aforementioned polynucleotide and a gRNA or a polynucleotide encoding a gRNA; or, comprises the aforementioned expression vector and a gRNA or a polynucleotide encoding a gRNA.

[0067] The system of the present invention can include one gRNA or multiple gRNAs at the same time. In some embodiments, 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 some embodiments, two or more guide RNAs target the same gene or transcript or locus. In some embodiments, two or more guide RNAs target different unrelated loci. In some embodiments, two or more guide RNAs target different but related loci.

[0068] In the present invention, the polynucleotide or the expression vector 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 specific embodiments, the polynucleotide or the expression vector comprises one or more modifications, so that more functions can be easily obtained, 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 specific embodiments, the DNA or RNA encoding the nuclease introduced into the cell is modified to edit any one or more genomic loci. The modification can be a single modification, or a combination of modifications.

[0069] In the present invention, the Cas9 nuclease, or the polynucleotide encoding the Cas9 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 cells, including stem cells, somatic cells, etc. The present invention is preferably used for mammalian cells HEK293T cells. The cells can be in vivo or in vitro.

[0070] In the gene editing system of the present invention, Cas9 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 gene editing system is a nucleic acid fragment (such as a DNA fragment) with a length of 20bp before the PAM sequence. In some embodiments, 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.

[0071] In the present invention, nuclease variants can also be formed by modification, mutation, DNA shuffling, etc., so that the nuclease variants have 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 the nuclease with a homologous or heterologous cleavage domain from a different nuclease (e.g., the HNH domain of a CRISPR-associated nuclease); by any modification method of DNA binding and / or DNA modification protein known in the art, such as methylation, demethylation, acetylation, etc., for example, the DNA targeting of the aforementioned nuclease can be changed. The DNA shuffling refers to exchanging sequence fragments between DNA sequences of nucleases from different sources to produce a chimeric DNA sequence encoding a synthetic protein with RNA-guided endonuclease activity. The modification, mutation, DNA shuffling, etc. can be used alone or in combination.

[0072] The present invention also provides a composition comprising one or more of the aforementioned Cas9 nuclease, the aforementioned polynucleotide, the aforementioned expression vector 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, it may also contain other low molecular weight polypeptides; 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.

[0073] The present invention also provides a cell, which contains the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned gene editing system.

[0074] In some embodiments, the cell is selected from a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be selected from Escherichia coli, Salmonella typhimurium, 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 johnsii, Bacteroides thaliana, Lactobacillus rhamnosus, Bacteroides massiliense, Parabacteroides feces, Fusobacterium death, Bifidobacterium breve or Listeria; or, the eukaryotic cell can be selected from yeast cells, Streptomyces, Drosophila S2 or Sf9 insect cells, CHO, COS.293 cells, or Bowes melanoma cells.

[0075] The present invention also provides a method for preparing the aforementioned Cas9 nuclease, which comprises: introducing the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned gene editing system into an engineered cell, inducing the expression of the exogenous gene, and separating and purifying to obtain the Cas9 nuclease.

[0076] In some embodiments, the engineered cell is selected from a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be selected from Escherichia coli, Salmonella typhimurium, 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 johnsii, Bacteroides thaliana, Lactobacillus rhamnosus, Bacteroides massiliense, Parabacteroides feces, Fusobacterium death, Bifidobacterium breve or Listeria; or, the eukaryotic cell can be selected from yeast cells, Streptomyces, Drosophila S2 or Sf9 insect cells, CHO, COS.293 cells, or Bowes melanoma cells.

[0077] In some specific embodiments, the introduction method can be selected from one or more of 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, and nanoparticle-mediated nucleic acid delivery.

[0078] The present invention also provides the use of the aforementioned Cas9 nuclease, the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned gene editing system in performing gene editing on a target gene in an in vitro cell or a cell-free environment or in preparing a gene editing tool.

[0079] 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.

[0080] 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; or, manipulating the expression of the target gene; or, genetically modifying the target gene; or, genetically modifying the target gene-related polypeptide; or, for intentional and controlled damage at any desired position of the target gene; or, for one or more of intentional and controlled repair at any desired position of the target gene.

[0081] In some specific embodiments, the target gene is a target DNA. In some embodiments, the target DNA can 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 aforementioned nuclease and gRNA, and the gRNA provides target specificity for the targeting complex by comprising a nucleotide sequence complementary to the target DNA; the aforementioned Cas9 nuclease provides site-specific editing 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.

[0082] The present invention also provides a gene editing method for in vivo or in vitro disease diagnosis or treatment purposes or non-disease diagnosis or treatment purposes, wherein the gene editing method is: contacting the target gene with the aforementioned gene editing system to achieve editing of the target gene. The gene editing method for in vitro or non-disease diagnosis or treatment purposes may be a gene editing method for industrial production purposes or a gene editing method for scientific research purposes.

[0083] In some embodiments, the gene editing method comprises the following steps:

[0084] a. introducing the aforementioned Cas9 nuclease, the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned gene editing system into an in vitro cell or a cell-free system;

[0085] b. introducing gRNA, also known as sgRNA, or a nucleic acid (e.g., DNA) suitable for in situ production of such gRNA;

[0086] c. contacting a cell or a target gene with the aforementioned nuclease, the aforementioned polynucleotide, the aforementioned expression vector 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 nuclease is guided to the target gene by its processed or unprocessed form of the gRNA.

[0087] In some specific embodiments, based on the efficiency of wild-type Cas9 nuclease in producing DNA deletions, the gene editing method is a method in which the aforementioned Cas9 nuclease cuts the DNA double strands to produce protruding broken ends, so as to produce a larger proportion of DNA deletions and a smaller proportion of DNA insertions.

[0088] In the present invention, the term "filling and ligation" generally means that the protruding broken ends are first filled into blunt ends by adding bases complementary to the protruding ends through base complementary pairing and then ligated.

[0089] 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.

[0090] In the present invention, the terms "peptide", "polypeptide" and "protein" are used interchangeably in the present invention 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.

[0091] In the present invention, 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.

[0092] In the present invention, 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 represent 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.

[0093] 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.

[0094] 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.

[0095] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both 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 generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record 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 realize the present invention.

[0096] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the literature, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; these series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0097] The information of the nucleotide sequence or amino acid sequence used in this application is as follows:

[0098] SEQ ID No.1

[0099] MAPKKKRKVG IHGVPAAMDK KYSIGLDIGT NSVGWAVITD EYKVPSKKFK VLGNTDRHSIKKNLIGALLF DSGETAEATR LKRTARRRYT RRKNRICYLQ EIFSNEMAKV DDSFFHRLEE SFLVEEDKKHERHPIFGNIV DEVAYHEKYP TIYHLRKKLV DSTDKADLRL IYLALAHMIK FRGHFLIEGD LNPDNSDVDKLFIQLVQTYN QLFEENPINA SGVDAKAILS ARLSKSRRLE NLIAQLPGEK KNGLFGNLIA LSLGLTPNFKSNFDLAEDAK LQLSKDTYDD DLDNLLAQIG DQYADLFLAA KNLSDAILLS DILRVNTEIT KAPLSASMIKRYDEHHQDLT LLKALVRQQL PEKYKEIFFD QSKNGYAGYI DGGASQEEFY KFIKPILEKM DGTEELLVKLNREDLLRKQR TFDNGSIPHQ IHLGELHAIL RRQEDFYPFL KDNREKIEKI LTFRIPYYVG PLARGNSRFAWMTRKSEETI TPWNFEEVVD KGASAQSFIE RMTNFDKNLP NEKVLPKHSL LYEYFTVYNE LTKVKYVTEGMRKPAFLSGE QKKAIVDLLF KTNRKVTVKQ LKEDYFKKIE CFDSVEISGV EDRFNASLGT YHDLLKIIKDKDFLDNEENE DILEDIVLTL TLFEDREMIE ERLKTYAHLF DDKVMKQLKR RRYTGWGRLS RKLINGIRDKQSGKTILDFL KSDGFANRNF MQLIHDDSLT FKEDIQKAQV SGQGDSLHEH IANLAGSPAI KKGILQTVKVVDELVKVMGR HKPENIVIEM ARENQTTQKG QKNSRERMKR IEEGIKELGS QILKEHPVEN TQLQNEKLYLYYLQNGRDMY VDQELDINRL SDYDVDHIVP QSFLKDDSID NKVLTRSDKN RGKSDNVPSE EVVKKMKNYWRQLLNAKLIT QRKFDNLTKA<h2 style=";text-align:left;direction:ltr">ERGGLSELDK AFFIKRQLVE TRQITKHVAQ ILDSRMNTKY DENDKLIREVKVITLKSKLV SDFRKDFQFY KVAEINNYHH AHDAYLNAVV GTALIKKYPK LESEFVYGDY KVYDVRKMIAKSEQEIGKAT AKYFFYSNIM NFFKTEITLA NGEIRKRPLI ETNGETGEIV WDKGRDFATV RKVLSMPQVNIVKKTEVQTG GFSKESILPK RNSDKLIARK KDWDPKKYGG FDSPTVAYSV LVVAKVEKGK SKKLKSVKELLGITIMERSS FEKNPIDFLE AKGYKEVKKD LIIKLPKYSL FELENGRKRM LASAGELQKG NELALPSKYVNFLYLASHYE KLKGSPEDNE QKQLFVEQHK HYLDEIIEQI SEFSKRVILA DANLDKVLSA YNKHRDKPIREQAENIIHLF TLTNLGAPAA FKYFDTTIDR KRYTSTKEVL DATLIHQSIT GLYETRIDLS QLGGDKRPAATKKAGQAKKKK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0100] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0101] MAPKKKRKVG IHGVPAAMDK KYSIGLDIGT NSVGWAVITD EYKVPSKKFK VLGNTDRHSIKKNLIGALLF DSGETAEATR LKRTARRRYT RRKNRICYLQ EIFSNEMAKV DDSFFHRLEE SFLVEEDKKHERHPIFGNIV DEVAYHEKYP TIYHLRKKLV DSTDKADLRL IYLALAHMIK FRGHFLIEGD LNPDNSDVDKLFIQLVQTYN QLFEENPINA SGVDAKAILS ARLSKSRRLE NLIAQLPGEK KNGLFGNLIA LSLGLTPNFKSNFDLAEDAK LQLSKDTYDD DLDNLLAQIG DQYADLFLAA KNLSDAILLS DILRVNTEIT KAPLSASMIKRYDEHHQDLT LLKALVRQQL PEKYKEIFFD QSKNGYAGYI DGGASQEEFY KFIKPILEKM DGTEELLVKLNREDLLRKQR TFDNGSIPHQ IHLGELHAIL RRQEDFYPFL KDNREKIEKI LTFRIPYYVG PLARGNSRFAWMTRKSEETI TPWNFEEVVD KGASAQSFIE RMTNFDKNLP NEKVLPKHSL LYEYFTVYNE LTKVKYVTEGMRKPAFLSGE QKKAIVDLLF KTNRKVTVKQ LKEDYFKKIE CFDSVEISGV EDRFNASLGT YHDLLKIIKDKDFLDNEENE DILEDIVLTL TLFEDREMIE ERLKTYAHLF DDKVMKQLKR RRYTGWGRLS RKLINGIRDKQSGKTILDFL KSDGFANRNF MQLIHDDSLT FKEDIQKAQV SGQGDSLHEH IANLAGSPAI KKGILQTVKVVDELVKVMGR HKPENIVIEM ARENQTTQKG QKNSRERMKR IEEGIKELGS QILKEHPVEN TQLQNEKLYLYYLQNGRDMY VDQELDINRL SDYDVDHIVP QSFLKDDSID NKVLTRSDKN RGKSDNVPSE EVVKKMKNYWRQLLNAKLIT QRKFDNLTKA<h2 style=";text-align:left;direction:ltr">ERGGLSELDK AGFIKRQLVE TRQITKHVAQ ILDSRMNTKY DENDKLIREVKVITLKSKLV SDFRKDFQFY KVREINNYHH AHDAYLNAVV GTALIKKYPK LESEFVYGDY KVYDVRKMIAKSEQEIGKAT AKYFFYSNIM NFFKTEITLA NGEIRKRPLI ETNGETGEIV WDKGRDFATV RKVLSMPQVNIVKKTEVQTG GFSKESILPK RNSDKLIARK KDWDPKKYGG FDSPTVAYSV LVVAKVEKGK SKKLKSVKELLGITIMERSS FEKNPIDFLE AKGYKEVKKD LIIKLPKYSL FELENGRKRM LASAGELQKG NELALPSKYVNFLYLASHYE KLKGSPEDNE QKQLFVEQHK HYLDEIIEQI SEFSKRVILA DANLDKVLSA YNKHRDKPIREQAENIIHLF TLTNLGAPAA FKYFDTTIDR KRYTSTKEVL DATLIHQSIT GLYETRIDLSQLGGDKRPAATKKAGQAKKKK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0102] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0103] atggccccaa agagaagcg gaaggtcggt atccacggtg tcccagcagc catggacagaagtactcca ttgggctcga tatcggcaca aacagcgtg gctggccgt cattacggac gagtacaggtgccgagca aaaattcaa gttcagatcctcgg accacacgca ataaattcaa tcattggcgccctcctgttc gactccgggg agacggccga agccacgcgg ctcaaagaa cagcacggcg cagatatacccgcagaaaga atcggatctg ctacctgcag gagatcttta gtaatgagat ggctaggtg gatgactctctcttccatag gctcacc taggatggagg tcaggagg gagcgccacc caatctttggcaatcgtg gacgaggtgg cgtaccatga aaagtacca accatatatc atctgaggaa gaagcttgtagacagtactg ataggctga cttgcggttg atctatctcg cgctggcgca tatgatcaa ttcggggacactccagacgaggtccca tgtcgacaaa ctctttacc aactggttcagacttacaat cagcttcg agagaaccc gatcacgca tccggagttg acgccaagc aatcctgacgctaggctgt ccaatcccg gcggtacgact aacctcatcg aactgtcagaccgacc tggcggacc ctgtcactcg ggctgacccc siactttaaa tctactcg acctggccgagatgccaag cttcaactga gcaagacac ctgtgat gatctcgaca atctgctggc ccagatcggcgaccagtacg cagacctttttttggcggca aagaacctgt cagacgccat tctgctgagt gatattctgcgagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag cgctatgatg agcaccaccaagacttgact ttgctgaagg cccttgtcag acagcaactg cctgagaagt acaaggaaat tttcttcgatcagtctaaaa atggctacgc cggatacatt gacggcggag caagccagga ggaattttac aaatttattaagcccatctt ggaaaaaatg gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcgcaaacagcgc actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatactcaggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc ctcacatttcggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg tggatgactc gcaaatcagaagagaccatc actccctgga acttcgagga agtcgtggat aagggggcct ctgcccagtc cttcatcgaaaggatgacta actttgataa aaatctgcct aacgaaaagg tgcttcctaa acactctctg ctgtacgagtacttcacagt ttataacgag ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcctgtctggagag cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacagctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg gaggatcgcttcaacgcatc cctgggaacg tatcacgatctcctgaaaat cattaaagac aaggactcc tggacaatgaggagaacgag gatacttg aggacattgt cctcaccctt acgttgttg agataggga gatgattgaacgcttga aaacttacgc tcatctctc ggacgacaggct tcatgaacag gccgaggcaggg agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcctggattctt aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctcacctttaggagg acatccagaa agcacagtt tctggccagg gggacaggcagtact tcacctact cccagctatc aaaaagggaa tactgcagac cgttaaggtac gtggatgaac tcgtcaagtaatgggaagg cataagcccg agaatacgt tatcgagatg gcccgagaga accaaactac ccagaagggacagaccagaca gtagggaaag gatgagagagg attagagg att caatccttaaggacaccc agttgaaac acccagcttc agaatgagaa gcttacctg tactacctgc agaacggcagggacatgtac gtggatcagg aactgacat caatcggctc tccgactacg acgtggatca tatcgtgccagtctgactgattgattcagtcatc cgataaaaat agagggagtgataacgt cccctcagaa gaagttgtca agaaaatgaaaattattgg cggcagctgc tgaacgccaactgatcaca caacggaagt tcgataatct gactaaggctgaacgaggtg gcctgtctga gttggataaagcattcttca tcaaaaggca gcttgttgag acacgccaga tcaccaagca cgtggcccaa attctcgattcacgcatgaa caccaagtac gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtctaagctggttc tcagatttca gaaggactt tcagttttat aaggtggccg agatcaacaa ttaccaccatgcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag cttgaatctgaatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca aagtctgagc aggaaataggcaaggccacc gctaagtact tcttttacag caatattattca agaccgagat tacactggccaatggagaga ttcggaagcg accacttatc gaaaacg gagaaag agaaatcgtg tgggacaagggtagggattt cgcgacagtc cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaa agaccgaagtacagaccgga ggcttctcca aggaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaaaaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta ctggttgtggccaaagtgga gaagggaag tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca caatcatggagcgatcaagc ttcgaaaaa accccatcga ctttctcgag gcgaaaggat ataaagaggt caaaaaagacctcatcatta agcttcccaa gtactctctc tttgagcttg aaaacggccggaaacgaatg ctcgctagtgcgggcgagct gcagaaaggt aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccagccactatgaa aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaacactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc gacgctaacctcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg gagcaggcag aaaacattatccacttgttt actctgacca acttgggcgc gcctgcagcc ttcaagtact tcgacaccac catagacagaaagcggtaca cctctacaaa ggaggtcctg gacgccacac tgattcatca gtcaattacg gggctctatgaaacaagaat cgacctctct cagctcggtg gagacaagcg tcctgctgct actaagaaag ctggtcaagctaagaaaaag aaaaaaa

[0104] SEQ ID No.4

[0105] atggccccaa agagaagcg gaaggtcggt atccacggtg tcccagcagc catggacagaagtactcca ttgggctcga tatcggcaca aacagcgtg gctggccgt cattacggac gagtacaggtgccgagca aaaattcaa gttcagatcctcgg accacacgca ataaattcaa tcattggcgccctcctgttc gactccgggg agacggccga agccacgcgg ctcaaagaa cagcacggcg cagatatacccgcagaaaga atcggatctg ctacctgcag gagatcttta gtaatgagat ggctaggtg gatgactctctcttccatag gctcacc taggatggagg tcaggagg gagcgccacc caatctttggcaatcgtg gacgaggtgg cgtaccatga aaagtacca accatatatc atctgaggaa gaagcttgtagacagtactg ataggctga cttgcggttg atctatctcg cgctggcgca tatgatcaa ttcggggacactccagacgaggtccca tgtcgacaaa ctctttacc aactggttcagacttacaat cagcttcg agagaaccc gatcacgca tccggagttg acgccaagc aatcctgacgctaggctgt ccaatcccg gcggtacgact aacctcatcg aactgtcagaccgacc tggcggacc ctgtcactcg ggctgacccc siactttaaa tctactcg acctggccgagatgccaag cttcaactga gcaagacac ctgtgat gatctcgaca atctgctggc ccagatcggcgaccagtacg cagacctttttttggcggca aagaacctgt cagacgccat tctgctgagt gatattctgcgagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag cgctatgatg agcaccaccaagacttgact ttgctgaagg cccttgtcag acagcaactg cctgagaagt acaaggaaat tttcttcgatcagtctaaaa atggctacgc cggatacatt gacggcggag caagccagga ggaattttac aaatttattaagcccatctt ggaaaaaatg gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcgcaaacagcgc actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatactcaggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc ctcacatttcggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg tggatgactc gcaaatcagaagagaccatc actccctgga acttcgagga agtcgtggat aagggggcct ctgcccagtc cttcatcgaaaggatgacta actttgataa aaatctgcct aacgaaaagg tgcttcctaa acactctctg ctgtacgagtacttcacagt ttataacgag ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcctgtctggagag cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacagctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg gaggatcgcttcaacgcatc cctgggaacg tatcacgatctcctgaaaat cattaaagac aaggactcc tggacaatgaggagaacgag gatacttg aggacattgt cctcaccctt acgttgttg agataggga gatgattgaacgcttga aaacttacgc tcatctctc ggacgacaggct tcatgaacag gccgaggcaggg agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcctggattctt aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctcacctttaggagg acatccagaa agcacagtt tctggccagg gggacaggcagtact tcacctact cccagctatc aaaaagggaa tactgcagac cgttaaggtac gtggatgaac tcgtcaagtaatgggaagg cataagcccg agaatacgt tatcgagatg gcccgagaga accaaactac ccagaagggacagaccagaca gtagggaaag gatgagagagg attagagg att caatccttaaggacaccc agttgaaac acccagcttc agaatgagaa gcttacctg tactacctgc agaacggcagggacatgtac gtggatcagg aactgacat caatcggctc tccgactacg acgtggatca tatcgtgccagtctgactgattgattcagtcatc cgataaaaat agagggagtgataacgt cccctcagaa gaagttgtca agaaaatgaaaattattgg cggcagctgc tgaacgccaactgatcaca caacggaagt tcgataatct gactaaggctgaacgaggtg gcctgtctga gttggataaagcaggcttca tcaaaaggca gcttgttgag acacgccaga tcaccaagca cgtggcccaa attctcgattcacgcatgaa caccaagtac gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtctaagctggttc tcagatttca gaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccatgcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag cttgaatctgaatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca aagtctgagc aggaaataggcaaggccacc gctaagtact tcttttacag caatattattca agaccgagat tacactggccaatggagaga ttcggaagcg accacttatc gaaaacg gagaaag agaaatcgtg tgggacaagggtagggattt cgcgacagtc cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaa agaccgaagtacagaccgga ggcttctcca aggaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaaaaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta ctggttgtggccaaagtgga gaagggaag tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca caatcatggagcgatcaagc ttcgaaaaa accccatcga ctttctcgag gcgaaaggat ataaagaggt caaaaaagacctcatcatta agcttcccaa gtactctctc tttgagcttg aaaacggccggaaacgaatg ctcgctagtgcgggcgagct gcagaaaggt aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccagccactatgaa aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaacactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc gacgctaacctcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg gagcaggcag aaaacattatccacttgttt actctgacca acttgggcgc gcctgcagcc ttcaagtact tcgacaccac catagacagaaagcggtaca cctctacaaa ggaggtcctg gacgccacac tgattcatca gtcaattacg gggctctatgaaacaagaat cgacctctct cagctcggtg gagacaagcg tcctgctgct actaagaaag ctggtcaagctaagaaaaag aaataa

[0106] Example 1 Identification of Cas9 nuclease DE Cas9 cleavage pattern

[0107] 1. Construction of Cas9 mutants

[0108] Cas9 mutagenesis primers:

[0109] DE Cas9-F: TTATAAGGTGGCCGAGATCAACAATTACCAC (SEQ ID NO.5);

[0110] DE Cas9-R:AACTGAAAGTCCTTTCTG (SEQ ID NO.6);

[0111] 1) Use the NEB mutation kit (Q5 Site-Directed Mutagenesis Kit, #E0554S) to construct a Cas9 mutant. First, use the Cas9 mutant G915F as a template for PCR amplification. The reaction is as follows:

[0112]

[0113]

[0114] Reaction conditions: 98°C, 30 sec;

[0115] 98℃,10sec

[0116] 60℃,20sec

[0117] 72℃,6.5min

[0118] 25 cycles;

[0119] 72°C, 3 min;

[0120] 2) KLD (Kinase, Ligase & DpnI) treatment, the reaction is as follows:

[0121]

[0122] Reaction conditions: room temperature for 10 minutes

[0123] 3) All the reaction products in 2) were used to transform competent bacteria Stbl3, and cultured overnight on LB plates containing ampicillin (Amp, 100 mg / L) at 37° C. Single clones were picked, and the plasmids were extracted and sent for sequencing, wherein the correct nucleotide sequence of nuclease DECas9 is shown in SEQ ID No. 3.

[0124] In addition, SpCas9 was randomly mutated according to the above construction method to obtain mutants E766A+R780A, E766A+G915F, E766A+F916P, E766A+△F916, E766A+R919P, E766A+Q920P, E766A+R976A, G915F+F916P, G915F+△F916, G915F+ R919P, G915F+Q920P, F916P+R919P, F916P+Q920P, F916P+R976A, ΔF916+R919P, ΔF916+Q920P, ΔF916+R976A, R919P+Q920P, R919P+R976A, Q920P-R976A were used as controls, and the sequences of these control mutants were all different from the DE Cas9 of the present invention.

[0125] 2. Cas9 nuclease mutants for DNA editing

[0126] (1) Construct sgRNAs targeting ATN1.

[0127] The sgRNAs target sequences:

[0128] ATN1 sgRNA4:GGTGATGTGTTGAGACTGGT (SEQ ID NO.7);

[0129] ATN1 sgRNA5:GATGTGTTGAGACTGGTGGG (SEQ ID NO.8);

[0130] The sgRNAs targeting sequences for ATN1 were purchased from Sangon Biotech (Shanghai) Co., Ltd., with forward and reverse deoxy oligonucleotides having 5' overhang ends "ACCG" and "AAAC" that can complement each other.

[0131] (2) Obtaining complementary double-stranded DNA with hanging ends

[0132] 1) Dissolve the oligonucleotide to 100 μM in ddH2O and dilute to 20 μM;

[0133] 2) Add forward and reverse deoxy oligonucleotides to the following reaction system:

[0134]

[0135] Reaction conditions: 95℃ water bath for 5min, then open the lid of the water bath to lower the temperature to about 60℃, cover with lid and cool to room temperature.

[0136] (3) Enzyme digestion of pGL3-U6-sgRNA-PGK-Puro vector

[0137] 1) The vector plasmid was digested with BsaI restriction endonuclease, and the reaction system was as follows:

[0138]

[0139] Reaction conditions: 37°C, 1.5 hours;

[0140] 2) Gel recovery and purification of DNA fragments according to the instructions of the gel recovery kit (Axygen).

[0141] (4) Ligation of the digested vector and the double-stranded DNA with hanging ends

[0142] The connection system is as follows:

[0143]

[0144]

[0145] Reaction conditions: room temperature for 1.5 hours;

[0146] (5) Transformation of ligation products

[0147] The ligation product was transformed with Stbl3 competent cell and cultured overnight at 37°C on LB plates containing ampicillin (Amp, 100 mg / L).

[0148] (6) Picking single clones for sequencing

[0149] 1) Pick a single colony from the ampicillin antibiotic LB plate and culture it in LB (Amp, 100 mg / L) liquid overnight;

[0150] 2) Plasmid extraction: follow the instructions of the plasmid extraction kit (Axygen);

[0151] 3) The extracted plasmid was sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0152] (7) Sequencing successful plasmids are extracted

[0153] 1) The plasmid successfully sequenced was re-transformed with Stbl3 competent medium and cultured overnight on LB plates containing Amp (100 mg / L);

[0154] 2) In the morning, pick a single colony and culture it in 2 ml LB (Amp, 100 mg / L) liquid medium for 8 hours, then transfer it to 200 ml LB (Amp, 100 mg / L) liquid medium and culture it overnight;

[0155] 3) Collect bacteria and extract plasmids according to the instructions of the plasmid extraction kit (Qiagen).

[0156] (8) Cell transfection using Lipofectamine 3000

[0157] 1) HEK293T cells were cultured in a culture flask at 37°C in a cell culture incubator containing 5% CO2. When the cells grew to 80-90% of the flask, the grown cells were plated in a 12-well plate with DMEM complete antibiotic-free medium and cultured overnight.

[0158] 2) When the cells in the 12-well plate grew to 80-90%, the prepared Cas9 and Cas9 mutant plasmids (800 ng) and sgRNAs plasmids (600 ng each) were transfected into the cells using Lipofectamine 3000, with two replicates for each sample.

[0159] 3) Two days after transfection, cells were collected and genomic extraction kit ( The genome was extracted using Genomic DNA Purification kit (Promega).

[0160] (9) Preparation of high-throughput sequencing library

[0161] Primers are designed approximately 30 bp downstream of the precise ligation site where the junction is expected to be deleted in the DNA fragment, and then the 5' end of the primer is added with an Illumina sequencing adapter with a barcode. The upstream primer can be designed at a position away from the splicing site and added with an Illumina sequencing adapter. PCR amplification is performed, and then beads are used for purification. The DNA product is dissolved in water, mixed in equal amounts to form a library for high-throughput sequencing.

[0162] (10) High-throughput sequencing data processing

[0163] The above results are as follows: when single sgRNA and Cas9 nuclease are used to edit genomic DNA fragments, Cas9 nuclease cuts the double strands of genomic DNA under the mediation of sgRNA to produce two broken ends, and these broken ends (DSB) may produce Indels under the action of the cell repair system. Then, we detected the repair of DNA fragments, and then used high-throughput sequencing technology to detect the connection of DNA fragments. In addition to the precise connection (Joinedprecisely) that was consistent with expectations, there was a certain proportion of indels at the connection of DNA fragments, and the types of indels were analyzed.

[0164] The wild-type SpCas9 nuclease (referred to as Cas9WT, WT) and DE Cas9 and other Cas9 mutants E766A+R780A, E766A+G915F, E766A+F916P, E766A+△F916, E766A+R919P, E766A+Q920P, E766A+R976A, G915F+F916P, G915F+△F916, G915F+R919P, G915 F+Q920P, F916P+R919P, F916P+Q920P, F916P+R976A, △F916+R919P, △F916+Q920P, △F916+R976A, R919P+Q920P, R919P+R976A, Q920P+R976A edited the genomic ATN1 site under the mediation of sgRNA. High-throughput sequencing was used to detect the editing results of the site, and the proportion of editing results was counted ( Figure 1 and Figure 2 ), the statistical results are shown in Table 1:

[0165] Table 1: Proportion of Deletion in ATN1 Sites Mediated by sgRNA4 / sgRNA5

[0166]

[0167]

[0168] From the above, we can see that under the mediation of sgRNA, the deletion ratio of DSB mediated by DE Cas9 is much higher than that of WTCas9, indicating that when the DE Cas9 nuclease mutant cuts the DNA chain that is not complementary to the sgRNA, the proportion of MMEJ repair pathway triggered by cutting upstream of PAM is significantly increased. Therefore, we prefer DE Cas9 as the base deletion nuclease mutant. Compared with SpCas9 nuclease (Cas9WT), G915F nuclease mutant, and R976A nuclease mutant, the proportion of base deletion caused by DSB mediated by DE Cas9 nuclease mutant was significantly increased. In the sgRNA4 site, deletion accounted for 31.30% in Cas9 WT, 44.33% in G915F, 52.75% in R976A, and 56.68% in DE Cas9; in the sgRNA5 site, deletion accounted for 46.42% in Cas9 WT, 43.82% in G915F, 46.50% in R976A, and 73.60% in DECas9 ( Figure 3 and Figure 4 ).

[0169] In addition to the altered deletion ratio, the DE Cas9 nuclease mutant altered the cleavage and repair patterns of Cas9 ( Figure 5 and Figure 6 ), the changes in the repair results are shown in the figure. Detailed analysis of the data also revealed that the proportion of insertions in DNA editing indels was significantly reduced. Figure 7 and Figure 8 As shown in Table 2:

[0170] Table 2

[0171] Selected Cas9 nuclease sgRNA4 site insertion ratio sgRNA5 site insertion ratio WT Cas9 66.61％ 52.38％ G915F 52.20％ 53.75％ R976A 45.33％ 47.15％ DE Cas9 30.03％ 23.75％

[0172] From the above results, we can see that the proportion of Insertion in the sgRNA4 site is 66.61% in SpCas9 nuclease (Cas9WT), 52.20% in G915F nuclease mutant, and 45.33% in R976A nuclease mutant. In contrast, it is only 30.03% in DE Cas9 nuclease mutant. The proportion of Insertion in the sgRNA5 site is 52.38% in SpCas9 nuclease (Cas9WT), 53.75% in G915F nuclease mutant, and 47.15% in R976A nuclease mutant. In contrast, it is only 23.75% in DE Cas9 nuclease mutant. Therefore, we believe that under the cleavage of DE Cas9 nuclease mutant, the cleavage and repair mode of this site is more inclined to the deletion of DNA fragments rather than the repair result of base insertion. Therefore, the deletion of DNA fragments replaces the addition of bases and becomes the dominant factor in the editing results.

[0173] In summary, the Cas9 nuclease (DE Cas9) of the present invention has different repair results for cutting the target genomic DNA fragment compared with the wild-type Cas9 nuclease. The Cas9 nuclease (DE Cas9) of the present invention can achieve cutting of the target genomic DNA fragment at a specific position and produce a larger proportion of deletion, reduce insertion in the indel ratio, and thus achieve precise DNA fragment editing at a specific position.

[0174] 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 Cas9 nuclease, characterized in that The Cas9 nuclease comprises a polypeptide having an amino acid sequence such as SEQ ID No.

1.

2. An isolated polynucleotide, characterized in that The polynucleotide encodes the Cas9 nuclease as described in claim 1.

3. The polynucleotide according to claim 2, characterized in that The polynucleotide comprises a nucleic acid fragment whose nucleotide sequence is shown as SEQ ID No.

3.

4. An expression vector, characterized in that: The expression vector comprises the polynucleotide according to any one of claims 2 to 3 and a plasmid backbone.

5. A gene editing system, characterized in that: The gene editing system comprises the nuclease as described in claim 1, and gRNA or a nucleic acid fragment encoding gRNA; or, comprises the polynucleotide as described in any one of claims 2-3, and gRNA or a polynucleotide encoding gRNA; or, comprises the expression vector as described in claim 4, and gRNA or a polynucleotide encoding gRNA.

6. A cell, characterized in that The cell contains the polynucleotide according to any one of claims 2-3, the expression vector according to claim 4, or the gene editing system according to claim 5.

7. Use of the nuclease according to claim 1, the polynucleotide according to any one of claims 2-3, the expression vector according to claim 4, or the gene editing system according to claim 5 in editing the target gene in vitro or in a cell-free environment or in preparing a gene editing tool.

8. The use according to claim 7, characterized in that The function of the gene editing is selected from one or more of gene cutting, gene deletion, gene insertion, point mutation, transcription inhibition or transcription activation.

9. An in vitro gene editing method, characterized in that: The gene editing method comprises: contacting the target gene with the gene editing system as described in claim 5 to achieve editing of the target gene.

10. The gene editing method according to claim 9, characterized in that: Based on the efficiency of wild-type Cas9 nuclease in producing DNA deletion, the gene editing method is a method in which the Cas9 nuclease as described in claim 1 cuts the DNA double strand to produce protruding broken ends, so as to produce a larger proportion of DNA deletion and a smaller proportion of DNA insertion, and the amino acid sequence of the wild-type Cas9 nuclease is shown in SEQ ID No. 2.

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