Cas9 nuclease and application thereof

Abstract

The invention belongs to the technical field of biology, and provides Cas9 nuclease, a CRISPR / Cas system comprising the Cas9 nuclease and application of the CRISPR / Cas system, and the Cas9 nuclease has an amino acid sequence as shown in SEQ ID NO: 1. The invention also provides a related nucleic acid, an expression vector comprising the nucleic acid and a recombinant cell. The genome editing activity of the Cas9 nuclease in mammalian cells is verified, and the Cas9 nuclease provides more tools and effective site selections for the in-vivo gene editing application direction.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to a Cas9 nuclease, a CRISPR / Cas system comprising the same, and uses thereof. Background Art

[0002] As an acquired immune mechanism of prokaryotes, the CRISPR / Cas system has RNA-mediated nuclease activity. The first system discovered that can be used for gene editing is the Cas9 system mediated by crRNA and tracrRNA. The system uses Cascade proteins, mainly Cas1 and Cas2, to capture phage virus DNA or exogenous plasmid DNA, and inserts it into its own direct repeat sequence to form a CRISPR sequence. The CRISPR sequence is transcribed into pre-crRNA, which is processed and modified into crRNA to form RNP with Cas9, and has RNA-guided DNA endonuclease activity. When the bacteria are infected with the virus again, the invading DNA can be targeted for cutting. This process also requires the participation of tracrRNA (Trans-activating crRNA) and the presence of PAM for specific recognition. The CRISPR / Cas system is widely used in gene editing because of its RNA-mediated nuclease activity.

[0003] In nature, the CRISPR / Cas system (clustered regularly interspaced shortpalindromic repeats) exists in more than 40% of bacterial genomes and 70% of archaeal genomes and provides immune protection for them. CRISPR systems can be divided into class 1 and class 2 according to homology. Class 1 includes type I, type III and type IV, and class 2 includes type II, type V and type VI. The most obvious feature of class 2 is that it forms a complex with a single Cas (CRISPR-associated protein) protein and crRNA (CRISPR RNA), performs targeted cutting function, and is simpler to operate. Although the class 2 system has only a single effector protein, the different types of effector proteins that have been discovered have obvious differences in protein molecular weight, domain, crRNA, PAM (Protospacer-adjacent motif) preference and nucleic acid cutting mode, providing more flexible options for gene editing. For example, the well-known Cas9, Cas12, and Cas13a all belong to class 2.

[0004] As early as 1987, Nakata et al. (Ishino, Y., et al., Nucleotide SEQuence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol, 1987. 169 (12): p. 5429-33) found a group of 29nt repeat sequences downstream of the iap gene when studying the conversion mechanism of alkaline phosphatase isozyme in Escherichia coli. These repeat sequences were separated by 32nt non-repeat sequences. In the following decade, this repeat sequence was reported in more and more bacteria and archaea. In 2000, Mojica et al. (Mojica, FJ, et al., Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol, 2000. 36 (1): p. 244-6.) classified this spacer repeat sequence as a clustered repeat sequence family present in most bacteria and archaea. In 2007, Barrangou et al. (Barrangou, R., et al., CRISPR provides acquired resistance against viruses in prokaryotes. Science, 2007. 315 (5819): p. 1709-12) found in Streptococcus thermophilus (S. thermophilus) that the type II CRISPR system (CRISPR / Cas9) played an important role in the degradation of foreign nucleic acid infection in bacteria as a natural adaptive immunity. Among them, the CRISPR spacer sequence provides targeting specificity for recognizing nucleic acids, and the Cas protein is active in the process of degrading nucleic acids. In 2011, Deltcheva et al. (Deltcheva, E., et al., CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 2011. 471(7340): p. 602-7) further clarified that RNA complex, Cas9 protein and endogenous RNase III are important components of the CRISPR / Cas system to exert targeting effects.Until 2012, Jinek et al. (Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337 (6096): p. 816-21) developed a chimeric sgRNA to guide the Cas9 protein to cut specific sites in the experiment, further simplifying the gene editing system and applying the system to genome editing for the first time. In 2013, Feng Zhang et al. (Cong, L., et al., Multiplex genome engineering using CRISPR / Cas systems. Science, 2013. 339 (6121): p. 819-23) used the CRISPR / Cas9 system to achieve multiple editing in the mammalian genome. Since the function and structure, mechanism and conditions of the CRISPR / Cas9 system have been studied and familiarized, the system has been widely used in the field of life sciences and has achieved remarkable results. In 2020, Jennifer A. Doudna and Emmanuelle Charpentier were awarded the Nobel Prize in Chemistry for their outstanding contributions to the CRISPR-Cas9 gene editing tool.

[0005] CRISPR technology has developed rapidly and can be applied to gene editing in bacteria, archaea and eukaryotic cells, but it has also exposed many problems. The recognition of target sequences by the CRISPR / Cas system is heavily dependent on the presence of PAM sequences. The limitations of PAM greatly limit its application scope in cell editing and nucleic acid detection. Secondly, the problem of off-target rate, off-target effects caused by low specificity, large fragment deletion and complex gene recombination need to be paid attention to. Although the current engineered CRISPR / Cas system has significantly improved in specificity and has been applied to gene therapy, such as β-thalassemia and sickle cell anemia, its PAM limitations still restrict the scope of application. Currently, the commercialized Cas proteins mainly include SpCas9 restricted by the 3' end PAM NGG and LbCas12a / AsCas12a restricted by the 5' end PAM TTTN.

[0006] Therefore, in order to break the current patent barriers of commercial Cas9 in vivo genome editing, enrich the resources of genome editing tools, and enhance the potential for subsequent engineering transformation, it is particularly important to discover systems with in vivo genome editing activity. Summary of the invention

[0007] The object of the present invention is to provide a Cas9 nuclease, a CRISPR / Cas system comprising the same and uses thereof.

[0008] Therefore, in a first aspect, the present invention provides a Cas9 nuclease having an amino acid sequence as shown in SEQ ID NO:1.

[0009] In a second aspect, the present invention provides a CRISPR / Cas system, which comprises the Cas9 nuclease according to the first aspect of the present invention.

[0010] In a third aspect, the present invention provides an isolated nucleic acid, wherein the nucleic acid sequence encodes the nucleotide sequence of the Cas9 nuclease according to the first aspect of the present invention.

[0011] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid sequence according to the third aspect of the present invention.

[0012] In a fifth aspect, the present invention provides a recombinant cell comprising the expression vector according to the fourth aspect of the present invention.

[0013] In a sixth aspect, the present invention provides a kit comprising the expression vector according to the fourth aspect of the present invention or the recombinant cell according to the fifth aspect of the present invention.

[0014] In a seventh aspect, the present invention provides the use of the Cas9 nuclease, CRISPR / Cas system, nucleic acid, expression vector or recombinant cell according to the present invention in gene editing.

[0015] The genome editing activity of the Cas9 nuclease of the present invention in mammalian cells has been verified, providing more tools and effective site selection for in vivo gene editing applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention is described in detail with reference to the following drawings.

[0017] Figure 1 The results of chip detection of the PAM preference of the Cas9 nuclease of the present invention are shown.

[0018] Figure 2 The in vivo genome editing activity (dsDNA) of the Cas9 nuclease of the present invention is shown. As shown in the figure, the Cas9 nuclease of the present invention has in vivo activity at the AAVS1 site g1 / g2 / g5 / g6 (AAVS1F1 / R1); and has in vivo activity at the EMX1 site g1 / g2 / g3 / g4 / g5 / g6 / g7 / g8 / g9 / g10 / g11 (EMX1F / R).

[0019] Figure 3 The Cas9 nuclease system of the present invention and the SpCas9 in vitro dsDNA cleavage electrophoresis diagram at different temperatures (AAVS1F1 / R1) are shown. The Cas9 nuclease of the present invention has a better catalytic effect at a higher temperature. DETAILED DESCRIPTION

[0020] In this article, the terms "Crispr", "crispr" or "CRISPR" all refer to Clustered regularly interspaced short palindromic repeats. Whether the terms are uppercase, lowercase or with the first letter capitalized, they are all commonly used expressions in the art. Correspondingly, there are different expressions in the CRISPR / Cas system due to the capitalization of letters. In addition, when representing bases, unless otherwise specified, the bases represented by the letters N and V have the usual meanings in the art, that is, N represents random or arbitrary bases A, T, C or G, and V represents random or arbitrary bases A, C or G.

[0021] The Cas9 enzyme cuts at the target DNA target site, and the target site is usually determined in the following way: an RNA molecule called Crispr RNA (crRNA) uses part of its sequence to combine with an RNA molecule called tracrRNA through base pairing to form a chimeric RNA (tracrRNA / crRNA), and then uses another part of the crRNA sequence to base pair with the target DNA site. As a result, the chimeric RNA guides the Cas protein to bind to this target site for cutting. This chimeric RNA is also called a guide RNA.

[0022] In this article, nucleic acid sequences include DNA or RNA, and reference to a DNA sequence also includes reference to its corresponding RNA sequence, and vice versa. Those skilled in the art know how to convert between the two, and know whether to use a DNA sequence or an RNA sequence in a specific situation.

[0023] In this article, the reference to a nucleic acid sequence includes the sequence itself, its reverse complementary sequence, and the complementary double-stranded sequence formed by them. Those skilled in the art know how to obtain its reverse complementary sequence from a nucleic acid sequence. The function of a sequence referred to herein includes that the sequence itself has this function, or its reverse complementary sequence has this function. For example, the reference to a nucleic acid sequence encoding a protein such as a Cas12a nuclease includes the reference that the nucleic acid sequence encodes the protein, or that its reverse complementary sequence encodes the protein. In application, those skilled in the art will know to select the sequence itself or its reverse complementary sequence, or the double strand formed by them.

[0024] Therefore, herein, as long as it does not contradict the common knowledge in the art, reference to a nucleic acid sequence is equivalent to reference to any one or more of the corresponding DNA, RNA, DNA double strand, RNA double strand and DNA-RNA double strand.

[0025] The inventors discovered a new Cas9 nuclease in the Staphylococcus warneri strain and named it BEST14. The new protein was optimized based on human codons, and a genome editing plasmid was constructed. Effective system delivery was achieved through liposome transfection, and the editing activity of the new system in the genome was verified at the "Safe harbor gene" AAVS1 site in the human genome. Effective editing sites were screened, laying the foundation for later tool or system transformation based on the new system. The PAMNNGG of the Cas9 nuclease of the present invention complements the PAM preference gene target of SpCas9. The Cas9 nuclease of the present invention is 1054 amino acids in size, which is significantly smaller than SpCas9 (1368 amino acids), laying the foundation for the subsequent adoption of multiple delivery methods.

[0026] Therefore, the present invention provides a Cas9 nuclease, and a CRISPR / Cas system comprising the same and uses thereof.

[0027] In a first aspect, the present invention provides a Cas9 nuclease having an amino acid sequence as shown in SEQ ID NO:1.

[0028] In a second aspect, the present invention provides a CRISPR / Cas system, which comprises the Cas9 nuclease according to the first aspect of the present invention.

[0029] In some embodiments, the CRISPR / Cas system further comprises at least one of the following:

[0030] crRNA, tracrRNA, or chimeric RNA formed by crRNA and tracrRNA. These RNAs can help the CRISPR / Cas system to perform gene editing functions.

[0031] In a third aspect, the present invention provides an isolated nucleic acid encoding the nucleotide sequence of the Cas9 nuclease according to the first aspect of the present invention.

[0032] In some embodiments, the nucleic acid further includes crRNA, tracrRNA, or a chimeric RNA formed by crRNA and tracrRNA, or a DNA sequence corresponding to the above sequences.

[0033] In some embodiments, the nucleic acid is DNA or RNA.

[0034] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid according to the third aspect of the present invention. The above nucleic acid sequence is constructed with a vector to obtain an expression vector, which can express the corresponding Cas9 nuclease in the target cell, thereby performing corresponding gene editing in the target cell. Commonly used vectors can be plasmids, lentiviruses, etc., for example, pET 28a vectors, pMD19 vectors, etc.

[0035] In a fifth aspect, the present invention provides a recombinant cell comprising the expression vector described in the fourth aspect of the present invention. The expression vector is introduced into a cell to form a recombinant cell, and the corresponding Cas9 nuclease is expressed by the expression vector to achieve gene editing of the recombinant cell. In some embodiments, the recombinant cell can be a eukaryotic cell, such as a plant cell or an animal cell.

[0036] In a sixth aspect, the present invention provides a kit comprising the expression vector according to the fourth aspect of the present invention or the recombinant cell according to the fifth aspect of the present invention.

[0037] In the seventh aspect, the present invention provides the use of the Cas9 nuclease, CRISPR / Cas system, nucleic acid, expression vector or recombinant cell according to the present invention in gene editing, wherein the Cas9 nuclease is the Cas protease described in the first aspect of the present invention, the CRISPR / Cas system is the CRISPR / Cas system described in the second aspect of the present invention, the nucleic acid is the nucleic acid described in the third aspect of the present invention, the expression vector is the expression vector described in the fourth aspect of the present invention, and the recombinant cell is the recombinant cell described in the fifth aspect of the present invention. For example, the use can be cell gene editing, gene therapy, nucleic acid detection, high-throughput sequencing. In a preferred embodiment, the use is cell gene editing.

[0038] The embodiments of the present invention will be described in detail below in conjunction with examples. It will be appreciated by those skilled in the art that the following examples are illustrative only and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used that do not indicate manufacturers are conventional products that can be obtained commercially.

[0039] Example 1: Experiment of expressing and purifying Cas9 system effector protein

[0040] Before protein expression and purification, the physicochemical properties of the protein, including isoelectric point, relative molecular mass, extinction coefficient, etc., were analyzed based on the protein sequence (as shown in SEQ ID NO: 1) using the ProtParam tool provided by ExPasy (https: / / web.expasy.org / protparam / ) to adjust the purification process and buffer.

[0041] The plasmid was introduced into the competent cells BL21(DE3)(Takara) by heat shock transformation method, and 300μL of non-antibiotic medium was added for 60min. The plate (LB plate, kanamycin resistance) was cultured at 37°C overnight, and a single colony was selected for expansion. The protein-expressing BL21(DE3) cells were cultured in LB medium (supplemented with 50mg / L kanamycin) at 37°C until OD600 reached 0.6, and protein expression was induced by adding 0.5mM isopropyl β-D-thiogalactopyranoside (IPTG). BL21(DE3) cells were further cultured at 16°C overnight (low temperature induction). The cells were collected by centrifugation at 6000 rpm and 4°C for 10 min, and the collected cells were resuspended in 1g:20mL binding buffer (50mM Tris-HCl, pH7.8, 500mM NaCl, 5mM imidazole), and the cells were lysed by ultrasonic disruption. Lysozyme (10mg / mL) and PMSF (0.1M) were added at a volume ratio of 1:100 before ultrasonic disruption. The cells after ultrasonic disruption were centrifuged at 12000 rpm and 4°C for 60 min, and the supernatant was collected.

[0042] (1) Affinity chromatography. Because the target protein carries a His tag, the inventors first selected a Ni-NTA gravity column for affinity chromatography to purify the protein. Before use, the filler should be washed three times with water and once with binding buffer. The filler was combined with the bacterial supernatant for 30 minutes and fully shaken every 5 minutes to allow as many target proteins as possible to bind to the Ni on the filler. Collect the flow-through. Wash with 5% elution buffer (50mM Tris-HCl, pH7.8, 500mM NaCl, 500mM imidazole) and collect the washed components. Elute the target protein with 50% elution buffer. Collect the target protein. Rinse the filler with elution buffer and collect the components. All collected components were sampled for SDS-PAGE to confirm the purification efficiency and recovery efficiency of the target protein.

[0043] (2) Molecular sieve chromatography. Use a 50K ultrafiltration tube to concentrate the target protein component to a volume of <2 mL and filter with a 0.56 μm filter membrane. Use AKTA (Cytiva) to separate proteins of different molecular weights through HiLoad 16 / 600 Superdex 200 pg (Cytiva). Load the sample with a 2 mL sample loop and pass through the chromatography column in a low-salt buffer (30 mM phosphate, pH 7.0, 150 mM NaCl, 0.4 mM DTT) at a flow rate of 0.5 mL / min. The collection plate is continuously collected. Samples are taken from the collection tubes at all UV peaks for SDS-PAGE electrophoresis to confirm the target protein component.

[0044] (3) Ion exchange chromatography. The target protein fraction was supplemented with low salt buffer to 20 mL. Filtered with a 0.56 μm filter membrane, the protein was separated and purified by HiTrap Capto SP ImpRes (Cytiva) using AKTA. The target protein was loaded onto the column and gradient eluted with 50% high salt buffer (30 mM phosphate, pH 7.0, 1 M NaCl, 0.4 mM DTT). The collection plate was continuously collected. Samples were collected from each tube for SDS-PAGE electrophoresis to confirm the purification efficiency and recovery efficiency. For systems with poor purity (<90%), cation exchange was used for further purification. The main peak collection tube fraction was concentrated using a 50 kDa ultrafiltration tube to a volume of <3 mL, and supplemented with low salt buffer to 20 mL. The above process was repeated to reduce the NaCl content in the target protein fraction. The protein was separated and purified by HiTrap Capto Q ImpRes (Cytiva) using AKTA. The target protein was loaded onto the column and gradient eluted with 50% high salt buffer. The collection plate was continuously collected. The collected samples from each tube were subjected to SDS-PAGE electrophoresis to confirm the purification efficiency and recovery efficiency.

[0045] Use 50K ultrafiltration tube to concentrate protein, measure absorbance A280 (1Abs = 1mg / mL) with ELISA, divide by extinction coefficient to get the real protein concentration. Add 70% sterile glycerol at a volume ratio of 1:1 and store at -20°C.

[0046] Example 2: Experiment to identify Cas9 PAM sequence

[0047] The PAM of BEST14 nuclease was identified using the DocMF method published by the applicant (Li et al., "DNB-based on-chip motiffinding: A high-throughput method to profile different types of protein-DNA interactions." Science Advances 6.31(2020):eabb3350.). The 3' end PAM sequence identified by BEST14 was NNGG (e.g. Figure 1 ).

[0048] Table 1: Nucleotide sequences used in PAM identification.

[0049] BEST14 (gRNA scaffold) SEQ ID NO:2 DNA SEQ ID NO:3

[0050] Example 3: Mammalian cell genome editing experiment

[0051] (1) Human cell culture

[0052] The human embryonic kidney cell-derived cell line HEK293T was selected for in vivo editing activity testing.

[0053] The culture conditions were: DMEM medium (high glucose, Gibco) containing 10% fetal bovine serum (FBS, Gibco), 1% non-essential amino acids (NEAA, Gibco), and 1% glutamine (GlutaMAX, Gibco), 37°C, 5% CO 2 concentration.

[0054] (2) Plasmid preparation

[0055] For the editing of HEK293T cells, the inventors selected endogenous genes AAVS1 (gene bank ID: AC005782.1) and EMX1 (gene bank ID: 2016) for targeted cutting verification.

[0056] Table 2 PCR primers for AAVS1 gene targeted region

[0057]

[0058] The nucleotide sequence of the targeting region of AAVS1 (AAVS1F1 / R1) is shown in SEQ ID NO:8.

[0059] The nucleotide sequence of the targeting region of EMX1 (EMX1F / R) is shown in SEQ ID NO:9.

[0060] The backbone sequence of the BEST14 editing vector is shown in SEQ ID NO:10.

[0061] For different genes, targeting sites were designed based on the corresponding proteins (Table 3). Gene editing plasmids of the corresponding proteins were designed and synthesized. The nucleotide sequences were synthesized by Beijing Liuhe BGI Technology Co., Ltd.

[0062] Table 3 BEST14 targeting site information

[0063] BEST14 guide sequence SEQ ID NO: AAVS1g1 11 AAVS1g2 12 AAVS1g5 13 AAVS1g6 14 EMX1g1 15 EMX1g2 16 EMX1g3 17 EMX1g4 18 EMX1g5 19 EMX1g6 20 EMX1g7 21 EMX1g8 22 EMX1g9 23 EMX1g10 24 EMX1g11 25

[0064] The following steps are performed to complete intracellular plasmid delivery (transfection), genome editing, and activity verification experiments:

[0065] (a) Take 15 mL of LB liquid culture medium (sterilized in advance at high temperature and high pressure, room temperature), add 15 μL of 1000X Amp antibiotic, use a 10 μL pipette tip to pick up the punctured strain containing the target plasmid (Beijing Liuhe Huada), put it into the culture medium, and culture it at 37°C, 200 rpm for 12-16 h;

[0066] (b) centrifuging the cultured and amplified bacterial solution at 8000 rpm for 3 min and discarding the culture medium;

[0067] (c) Use an endotoxin-free kit (Tiangen Biochemical) to extract the target plasmid according to the instructions;

[0068] (d) After extraction, the DNA concentration was quantified using Nanodrop (Thermo) and stored at -20°C.

[0069] (3) Plasmid transfection

[0070] (a) One day before transfection, use a pipette to remove the original culture medium of HEK293T cells (~90% confluence) in the seed plate, slowly add about 2 mL of 37°C preheated DPBS (Gibco) along the wall to wash the cell surface, and then add 1 mL of preheated digestion solution (TrypLE Express, Gibco) to digest. After about 3 minutes, add an appropriate amount of preheated DMEM medium containing serum to terminate the digestion, resuspend by pipetting, take a small amount of cell suspension and gently pipette and mix with an equal proportion of trypan blue dye (Solarbio), take about 20 μL of the mixture and add it to a cell counting plate (Countstar) and use a cell analyzer (Countstar Rigel S2) to count the live cells. Finally, use a 12-well cell culture plate for plating and culture, with about 0.5 to 1×10 cells per well. 6 cells;

[0071] (b) When the confluence of the transfected cells reaches 50% to 70%, the cells can be transfected with the target plasmid using the Lipofectamine 3000 kit (Invitrogen) according to the instructions (2 μg of plasmid per well, 2.4 μL of Lipofectamine 3000 Reagent). The culture medium can be replaced as needed 6 hours after transfection;

[0072] (c) After transfection, cells need to be cultured for 2-3 days to allow sufficient gene editing;

[0073] (d) After the cell culture is completed, the transfection efficiency is calculated and the cells are recovered. The cell digestion and resuspension steps are the same as above (the amount of relevant reagents is added according to the proportion calculated by the cell culture area). After adding about 20 μL of cell suspension to the cell counting plate, use a cell analyzer and use the green fluorescence (GFP) counting channel to calculate the plasmid transfection efficiency. Finally, the remaining cells are transferred to a 1.5 mL centrifuge tube, centrifuged at 12000 rpm for 1 min, and the upper culture medium is removed to harvest the cells.

[0074] (4) Identification of genome editing activity

[0075] After harvesting the cells, genome extraction and T7E1 restriction digestion experiments were performed to initially detect editing. The steps are as follows:

[0076] (a) Genomic DNA extraction: Genomic DNA was extracted using a genomic DNA extraction kit (Tiangen), and the genomic (gDNA) concentration was quantified using Nanodrop and stored at -20°C.

[0077] (b) Targeted region PCR: A high-fidelity amplification enzyme (PrimeSTAR GXL DNA Polymerase, Takara) was used to amplify the target site region from gDNA. The amplification primers are shown in Table 2. The deoxynucleotide sequences used were synthesized at the Shenzhen National Gene Bank Synthesis and Editing Platform. After a clear and single target band was observed under a gel imager by 1% agarose gel (TAE) electrophoresis (7.5 V / cm, 30 min), the gel was cut and the target band was purified and recovered using a PCR purification and gel extraction kit (NucleoSpinExtract, MN). The concentration was measured using Nanodrop.

[0078] (c) Denaturation and annealing: The mutant DNA and control reaction system were mixed as shown in Table 4, and subjected to heating denaturation and annealing. The above program settings of the PCR instrument (Bio-rad) are shown in Table 5.

[0079] (d) T7E1 digestion: Add 0.3 μL of T7EI nuclease to the reaction system in step (c), totaling 20 μL of reaction system. Perform digestion reaction at 37°C for 20 min.

[0080] (e) Activity detection: After the reaction is completed, 4 μL of 6× Gel Loading Dye (NEB) is added and the bands are detected by agarose gel electrophoresis (the detailed steps are the same as the above agarose gel electrophoresis operation).

[0081] Table 4 Annealing reaction system

[0082]

[0083]

[0084] Table 5 Denaturation and annealing conditions

[0085] temperature time 95℃ 3min -2℃ / min 10min 75℃ 30sec -6℃ / min 8min 27℃

[0086] like Figure 2 As shown, the new BEST14 system has human cell editing activity and has in vivo activity in AAVS1 gene g1 / g2 / g5 / g6 and EMX1 gene g1 / g2 / g3 / g4 / g5 / g6 / g7 / g8 / g9 / g10 / g11.

[0087] Example 4 Comparison of in vitro catalytic activity of the novel protein and SpCas9 at different temperatures

[0088] Perform in vitro cleavage experiments using the gene editing system according to the following protocol:

[0089] 1. Using the editing plasmid shown in Example 3 as the target, amplify and obtain the transcription template, using MEGAshortscript TM Kit in vitro transcribes the corresponding gRNA, and the primers and templates are shown in Table 6. Use the primer pairs in Table 2 to amplify and prepare double-stranded DNA substrates, and determine that the DNA concentration is not less than 40ng / μL and the gRNA concentration is not less than 800ng / μL

[0090] 2. Mix 2 μL 10×NEB buffer 3.1, 2 μL 10-30 μM protein, and 1-2 μL gRNA, add RNase-free water to 16 μL, and shake to mix. Figure 3 After incubation at the specified temperature for 15 min, 4 μL of substrate DNA was added and reacted at the gradient temperature for 0.5 h.

[0091] 3. Take 10 μL of the product and perform electrophoresis on 1% agarose gel.

[0092] Table 6 gRNA transcription template amplification primers and sequences

[0093]

Claims

1. A Cas9 nuclease having an amino acid sequence as shown in SEQ ID NO:

1.

2. A CRISPR / Cas system comprising the Cas9 nuclease according to claim 1.

3. The CRISPR / Cas system according to claim 2, further comprising at least one of the following: crRNA, tracrRNA or a chimeric RNA formed by crRNA and tracrRNA.

4. An isolated nucleic acid having a nucleotide sequence encoding the Cas9 nuclease of claim 1.

5. The nucleic acid according to claim 4, wherein the nucleotide further comprises crRNA, tracrRNA, or a chimeric RNA formed by crRNA and tracrRNA, or a DNA sequence corresponding to the above sequence. The nucleic acid according to claim 4 or 5, wherein the nucleic acid is DNA or RNA.

7. An expression vector comprising the nucleic acid according to any one of claims 4 to 6. A recombinant cell comprising the expression vector according to claim 7.

9. A kit comprising the expression vector according to claim 7 or the recombinant cell according to claim 8.

10. Use of the Cas9 nuclease of claim 1, the CRISPR / Cas system of claim 2 or 3, the nucleic acid of any one of claims 4 to 6, the expression vector of claim 7 or the recombinant cell of claim 8 in gene editing, preferably, the use is cell gene editing, nucleic acid detection, and high-throughput sequencing.

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