Cas9 nuclease system and its applications

Abstract

The application belongs to the technical field of biology, and provides a Cas9 nuclease system and application thereof, the Cas9 nuclease system comprising a Cas9 nuclease with an amino acid sequence as shown in SEQ ID NO:1 and a gRNA, wherein the sequence of the gRNA scaffold is as shown in SEQ ID NO:3. The application also provides related nucleic acids, expression vectors comprising the nucleic acids and recombinant cells. The genome editing activity of the Cas9 nuclease system of the application in mammalian cells has been verified, which provides more tools and effective site selection for the application direction of in vivo gene editing.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a Cas9 nuclease system and its uses. Background Technology

[0002] The CRISPR / Cas system, as an acquired immune mechanism in prokaryotes, possesses RNA-mediated endonuclease activity. The earliest discovered system for gene editing was the Cas9 system, mediated by crRNA and tracrRNA. This system uses Cascade proteins, primarily Cas1 and Cas2, to capture bacteriophage viral DNA or exogenous plasmid DNA, inserting it into its own orthogonal repeat sequence to form a CRISPR sequence. The CRISPR sequence is transcribed into pre-crRNA, which is then processed and modified into crRNA to form an RNP with Cas9, possessing RNA-directed DNA endonuclease activity. When bacteria are reinfected by the virus, the invading DNA can be targeted and cleaved. This process also requires the participation of tracrRNA (trans-activating crRNA) and the presence of a specifically recognized PAM. The CRISPR / Cas system is widely used in gene editing due to its RNA-mediated endonuclease activity.

[0003] The CRISPR system can be divided into class 1 and class 2 based on homology. Class 1 includes type I, type III, and type IV, while class 2 includes type II, type V, and type VI. The most obvious characteristic of class 2 is that it consists of a single Cas (CRISPR-associated protein) protein forming a complex with crRNA (CRISPR RNA) to perform targeted cleavage, making it simpler to operate. Although the class 2 system only has a single effector protein, the different types of effector proteins discovered have significant differences in protein molecular weight, domains, crRNA, PAM (Protospacer-adjacent motif) preference, and nucleic acid cleavage 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 Seqence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichiacoli, and identification of the gene product. J Bacteriol, 1987, 169(12): p. 5429-33) discovered a set of 29 nt repetitive sequences downstream of the iap gene while studying the alkaline phosphatase isozyme conversion mechanism in Escherichia coli. These repetitive sequences were separated by 32 nt non-repetitive sequences. In the following decade, such repetitive sequences were reported to have been found in an increasing number of 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 spaced repeat sequence as a family of clustered repeat sequences found 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) discovered in Streptococcus thermophilus that the type II CRISPR system (CRISPR / Cas9) plays an important role as an innate adaptive immunity in the degradation of foreign nucleic acid infections in bacteria. Among them, the CRISPR spacer sequence provides target specificity for recognizing nucleic acids, and the Cas protein plays an active role in the degradation of 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 elucidated that the RNA complex, Cas9 protein, and endogenous RNase III are important components for the targeting function of the CRISPR / Cas system.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 experiments, further simplifying the gene editing system and applying it 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) achieved multiplex editing in the mammalian genome using the CRISPR / Cas9 system. Since the function, structure, mechanism, and conditions of the CRISPR / Cas9 system have been studied and understood, the system has been widely used in the 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 CRISPR / Cas system's recognition of target sequences heavily relies on the presence of PAM sequences, and the limitations of PAM significantly restrict its application in cell editing and nucleic acid detection. Secondly, the off-target rate, low specificity leading to off-target effects, as well as large fragment deletions and complex gene recombination, require further attention. Although engineered CRISPR / Cas systems have significantly improved specificity and have been applied to gene therapy, such as for β-thalassemia and sickle cell anemia, the limitations of PAM still restrict their applicability. Currently, commercially available Cas proteins mainly include SpCas9, which is restricted by the 3' PAM NGG, and LbCas12a / AsCas12a, which is restricted by the 5' PAM TNT.

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

[0007] The purpose of this invention is to provide a Cas9 nuclease system and its uses.

[0008] Therefore, in a first aspect, the present invention provides a Cas9 nuclease system comprising a Cas9 nuclease having an amino acid sequence as shown in SEQ ID NO: 1 and gRNA, wherein the sequence of the gRNA scaffold is shown in SEQ ID NO: 3.

[0009] In a second aspect, the present invention provides an isolated nucleic acid that encodes the nucleotide sequence of the Cas9 nuclease according to the first aspect of the present invention, and further includes gRNA, or a DNA sequence corresponding to the gRNA.

[0010] In a third aspect, the present invention provides an expression vector comprising the nucleic acid described in the second aspect of the present invention.

[0011] In a fourth aspect, the present invention provides a recombinant cell comprising the expression vector described in the third aspect of the present invention.

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

[0013] In a sixth aspect, the present invention provides the use of the Cas9 nuclease system, nucleic acid, expression vector or recombinant cell according to the invention in gene editing.

[0014] The genome editing activity of the Cas9 nuclease system of this invention in mammalian cells has been verified, providing more tools and effective site selection for in vivo gene editing applications. Attached Figure Description

[0015] The present invention will be described in detail with reference to the following figures.

[0016] Figure 1 The results of PAM preference for chip detection of the Cas9 nuclease of the present invention are shown.

[0017] Figure 2 The image shows an in vivo genome editing activity verification (dsDNA) electrophoresis diagram (AAVS1F4 / R4) of the Cas9 nuclease system of the present invention. As shown in the figure, g1 / g2 / g5 have significant activity.

[0018] Figure 3 The Cas9 nuclease system of the present invention and in vitro dsDNA cutting electrophoresis images of SpCas9 at different temperatures are shown (AAVS1F1 / R1). The Cas9 nuclease of the present invention exhibits excellent catalytic performance at the overall experimental temperature. Detailed Implementation

[0019] In this paper, the terms "Crispr," "crispr," or "CRISPR" all refer to clustered regularly interspaced short palindromic repeats. Whether the terminology is uppercase, lowercase, or begins with a capital letter, it represents the commonly used terminology in the field. Correspondingly, the CRISPR / Cas system uses different terminology due to capitalization. Furthermore, when representing bases, unless otherwise specified, the letters N and V represent bases with their usual meanings in the field: N represents a random or arbitrary base A, T, C, or G, and V represents a random or arbitrary base A, C, or G.

[0020] The Cas9 enzyme cuts at target DNA sites, typically by determining the target site in the following way: an RNA molecule called CRISPR RNA (crRNA) uses a portion of its sequence to bind with an RNA molecule called tracrRNA through base pairing, forming a chimeric RNA (tracrRNA / crRNA). Then, the other portion of the crRNA sequence pairs with the target DNA site. In this way, the chimeric RNA guides the Cas protein to bind to this target site for cleavage. This chimeric RNA is also called guide RNA.

[0021] In this document, 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 will understand how to convert between the two and will know whether to use a DNA sequence or an RNA sequence in a specific context.

[0022] In this document, references to nucleic acid sequences include the sequence itself, its inverse complementary sequence, and the complementary double-stranded sequence formed by them. Those skilled in the art will understand how to derive the inverse complementary sequence from a nucleic acid sequence. The function of a sequence referred to herein includes the sequence itself having that function, or its inverse complementary sequence having that function. For example, referring to a nucleic acid sequence encoding a protein such as the Cas12a nuclease includes referring to the nucleic acid sequence encoding that protein, or its inverse complementary sequence encoding that protein. In application, those skilled in the art will understand the importance of selecting the sequence itself, its inverse complementary sequence, or the double-stranded sequence formed by them.

[0023] Therefore, in this article, as long as it does not contradict common sense in the field, mentioning nucleic acid sequences is equivalent to mentioning any one or more of the corresponding DNA, RNA, DNA double strand, RNA double strand, and DNA-RNA double strand.

[0024] The inventors discovered a novel Cas9 nuclease in *Eubacterium sp.* strain and named it BEST12. Based on human codon optimization, a genome editing plasmid was constructed using the new protein. Efficient systemic delivery was achieved via liposome transfection. The editing activity of the new system was verified at the AAVS1 site of the human genome's "Safe harbor gene," and effective editing sites were screened, laying the foundation for future tool or system modifications based on this new system. The PAM of the Cas9 nuclease of this invention effectively expands the screening application range of the Cas9 system in AT-rich gene sequences. The Cas9 nuclease of this invention is 1112 amino acids in size, significantly smaller than SpCas9 (1368 amino acids), paving the way for subsequent use of various delivery methods.

[0025] Therefore, the present invention provides a Cas9 nuclease system and its uses.

[0026] In a first aspect, the present invention provides a Cas9 nuclease system comprising a Cas9 nuclease having an amino acid sequence as shown in SEQ ID NO: 1 and gRNA, wherein the sequence of the gRNA scaffold is shown in SEQ ID NO: 3.

[0027] In a second aspect, the present invention provides an isolated nucleic acid that encodes the nucleotide sequence of the Cas9 nuclease according to the first aspect of the present invention, and further includes gRNA, or a DNA sequence corresponding to the gRNA.

[0028] In some implementations, the nucleic acid is DNA or RNA.

[0029] In a third aspect, the present invention provides an expression vector comprising the nucleic acid described in the second aspect of the invention. The above-described nucleic acid is used to construct an expression vector, which can express the corresponding Cas9 nuclease in target cells, thereby enabling corresponding gene editing in the target cells. Commonly used vectors can be plasmids, lentiviruses, etc., such as pET 28a vector, pMD19 vector, etc.

[0030] In a fourth aspect, the present invention provides a recombinant cell comprising the expression vector described in the third aspect of the invention. By introducing the expression vector into the cell to form a recombinant cell, and by expressing the corresponding Cas9 nuclease using the expression vector, gene editing of the recombinant cell can be achieved. In some embodiments, the recombinant cell may be a eukaryotic cell, such as a plant cell or an animal cell.

[0031] In a fifth aspect, the present invention provides a kit comprising the expression vector according to the third aspect of the invention or the recombinant cells according to the fourth aspect of the invention.

[0032] In a sixth aspect, the present invention provides the use of the Cas9 nuclease system, nucleic acid, expression vector, or recombinant cell according to the present invention in gene editing, wherein the Cas9 nuclease system is the Cas9 nuclease system described in the first aspect of the present invention, the nucleic acid is the nucleic acid described in the second aspect of the present invention, the expression vector is the expression vector described in the third aspect of the present invention, and the recombinant cell is the recombinant cell described in the fourth aspect of the present invention. For example, the use may be cell gene editing, gene therapy, nucleic acid detection, or high-throughput sequencing. In a preferred embodiment, the use is cell gene editing.

[0033] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are merely illustrative and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0034] Example 1: Experiment on expression and purification of Cas9 system effector proteins

[0035] Before protein expression and purification, the physicochemical properties of the protein, including isoelectric point, relative molecular mass, and extinction coefficient, were analyzed using the ProtParam tool provided by ExPasy (https: / / web.expasy.org / protparam / ) based on the protein sequence (as shown in Table 1) in order to adjust the purification process and buffer.

[0036] The plasmid was introduced into competent BL21(DE3)(Takara) cells using a heat shock transformation method, and the cells were cultured in 300 μL of antibiotic-free medium for 60 min. The cells were then plated (LB plate, kanamycin resistant) and cultured overnight at 37°C. Single colonies were selected for expansion. BL21(DE3) cells expressing the protein were cultured in LB medium (supplemented with 50 mg / L kanamycin) at 37°C until the OD600 reached 0.6. Protein expression was induced by adding 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG). BL21(DE3) cells were then cultured overnight at 16°C (low-temperature induction). Collect bacterial cells by centrifugation at 6000 rpm and 4°C for 10 min. Resuspend the collected bacterial cells in a 1 g:20 mL binding buffer (50 mM Tris-HCl, pH 7.8, 500 mM NaCl, 5 mM imidazole). Lyse the cells by sonication. Before sonication, add lysozyme (10 mg / mL) and PMSF (0.1 M) at a volume ratio of 1:100. Centrifuge the sonicated bacterial cells at 12000 rpm and 4°C for 60 min, and collect the supernatant.

[0037] (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 packing material should be washed three times with water and once with binding buffer. The packing material is bound to the bacterial supernatant for 30 min, with thorough shaking every 5 min to allow as much of the target protein as possible to bind to the Ni on the packing material. Flow-through is collected. The material is washed with 5% elution buffer (50 mM Tris-HCl, pH 7.8, 500 mM NaCl, 500 mM imidazole), and the washed components are collected. The target protein is eluted with 50% elution buffer. The target protein is collected. The packing material is rinsed with elution buffer, and the fractions are collected. All collected fractions are sampled and subjected to SDS-PAGE to confirm the purification and recovery efficiency of the target protein.

[0038] (2) Molecular sieve chromatography. The target protein fraction was concentrated to a volume <2 mL using a 50K ultrafiltration tube and filtered through a 0.56 μm filter membrane. Proteins with different molecular weights were separated using AKTA (Cvtiva) via a HiLoad 16 / 600 Superdex 200 pg (Cytiva) column. Samples were loaded onto a 2 mL loop and passed through the column in 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. Continuous collection was performed. Samples from all UV peak collection tubes were taken for SDS-PAGE electrophoresis to confirm the target protein fraction.

[0039] (3) Ion exchange chromatography. Add low-salt buffer to 20 mL to obtain the target protein fraction. Filter using a 0.56 μm filter membrane and purify the protein using AKTA via HiTrap Capto SP ImpRes (Cytiva). Attach the target protein to the column and elute with a gradient of 50% high-salt buffer (30 mM phosphate, pH 7.0, 1 M NaCl, 0.4 mM DTT). Collect continuously. Perform SDS-PAGE electrophoresis on samples from each collected tube to confirm purification and recovery efficiency. For systems with poor purity (<90%), further purification is performed using cation exchange. Concentrate the main peak fraction in the collection tube to a volume <3 mL using a 50 kDa ultrafiltration tube, add low-salt buffer to 20 mL, and repeat the above process to reduce the NaCl content in the target protein fraction. Purify the protein using AKTA via HiTrap Capto Q ImpRes (Cytiva). Attach the target protein to the column and elute with a gradient of 50% high-salt buffer. Collect continuously. The collected samples were subjected to SDS-PAGE electrophoresis to confirm the purification and recovery efficiency.

[0040] Protein was concentrated using 50K ultrafiltration tubes, and the absorbance (A280) was measured using a microplate reader (1 Abs = 1 mg / mL). The absorbance was then divided by the extinction coefficient to obtain the true protein concentration. 70% sterile glycerol was added at a 1:1 volume ratio and stored at -20℃.

[0041] Table 1: Sequences of proteins expressed and purified in Example 1

[0042] Name SEQ ID NO: Wild type BEST12 sequence 1 BEST12 gene coding sequence 2

[0043] Example 2: Experiment for identifying Cas9 PAM sequences

[0044] The PAM of the BEST12 nuclease was identified using the applicant's published DocMF method (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.). Bioinformatics analysis revealed that the 3' PAM sequence of BEST12 was NYRRV (e.g., ...). Figure 1 ).

[0045] Table 2: Nucleic acid sequences used in PAM identification.

[0046] BEST12 (gRNA scaffold) SEQ ID NO: 3 DNA SEQ ID NO: 4

[0047] Example 3: Mammalian Cell Genome Editing Experiment

[0048] (1) Human cell culture

[0049] The human embryonic kidney cell-derived cell line HEK293T was selected for in vivo editing activity testing.

[0050] The culture conditions were as follows: DMEM medium (high glucose, Gibco) containing 10% fetal bovine serum (FBS, Gibco), 1% non-essential amino acids (NEAA, Gibco), and 1% glutamine (GlutaMAX, Gibco), at 37°C and 5% CO2 concentration.

[0051] (2) Plasmid preparation

[0052] For editing HEK293T cells, the inventors used the endogenous gene AAVS1 (gene bank ID: AC005782.1) for targeted cleavage verification.

[0053] Table 3 Primers for PCR amplification of the AAVS1 gene target region

[0054]

[0055] The nucleotide sequence of the target region of AAVS1 (AAVS1F1 / R1) is shown in SEQ ID NO: 9.

[0056] The nucleotide sequence of the target region of AAVS1 (AAVS1F4 / R4) is shown in SEQ ID NO: 10.

[0057] The BEST12 edit vector backbone sequence is shown in SEQ ID NO: 11.

[0058] Target sites were designed for different genes based on their corresponding proteins (Table 4). Gene editing plasmids for the corresponding proteins were designed and synthesized. All nucleotide sequences were synthesized at Beijing BGI Genomics Co., Ltd.

[0059] Table 4 BEST12 Target Site Information

[0060] BEST12 guide sequence SEQ ID NO: AAVS1g1 12 AAVS1g2 13 AAVS1g5 14

[0061] The following steps were used to complete the intracellular plasmid delivery (transfection), genome editing, and activity verification experiments:

[0062] (a) Take 15 mL of LB liquid medium (sterilized by high temperature and autoclave in advance, at room temperature), add 15 μL of 1000X Amp antibiotic, use a 10 μL pipette tip to pick up the stab strain (Beijing Liuhe Huada) that preserves the target plasmid, put it into the medium, and incubate at 37℃ and 200 rpm for 12-16 h.

[0063] (b) Centrifuge the cultured and amplified bacterial solution at 8000 rpm for 3 min and discard the culture medium;

[0064] (c) Extract the target plasmid using an endotoxin-free kit (Tiangen Biotech) according to the instructions;

[0065] (d) After extraction, the DNA concentration was quantified using Nanodrop (Thermo) and stored at -20℃.

[0066] (3) Plasmid transfection

[0067] (a) One day before transfection, the original culture medium of HEK293T cells (~90% confluence) in the seeding tray was aspirated using a pipette. Approximately 2 mL of pre-warmed 37°C DPBS (Gibco) was slowly added along the cell wall to wash the cell surface. Then, 1 mL of pre-warmed digestion buffer (TrypLE Express, Gibco) was added for digestion. After approximately 3 minutes, an appropriate amount of pre-warmed DMEM containing serum was added to terminate digestion. The cells were resuspended by pipetting. A small amount of the cell suspension was gently mixed with an equal proportion of trypan blue (Solarbio) by pipetting. Approximately 20 μL of the mixture was added to a Countstar cell counting chamber, and viable cell counts were performed using a Countstar Rigel S2 analyzer. Finally, cells were seeded in 12-well cell culture plates, with approximately 0.5–1 × 10⁶ cells per well. 6 One cell;

[0068] (b) When the confluence of cells to be transfected reaches 50%–70%, the target plasmid can be transfected using the Lipofectamine 3000 kit (Invitrogen) according to the instructions (2 μg plasmid and 2.4 μL Lipofectamine 3000 Reagent per well). Change the culture medium as needed 6 hours after transfection.

[0069] (c) After transfection, cells need to be cultured for 2-3 days to allow for sufficient gene editing;

[0070] (d) After cell culture, transfection efficiency was calculated and cells were recovered. Cell digestion and resuspension were performed as described above (reagent amounts were added proportionally based on the cell culture area). Approximately 20 μL of cell suspension was added to a cell counting chamber, and the plasmid transfection efficiency was calculated using a cell analyzer and the green fluorescence (GFP) counting channel. Finally, the remaining cells were transferred to 1.5 mL centrifuge tubes, centrifuged at 12000 rpm for 1 min, and the supernatant was removed to harvest the cells.

[0071] (4) Identification of genome editing activity

[0072] After harvesting cells, preliminary detection and editing were performed using genome extraction and T7E1 restriction enzyme digestion experiments, as follows:

[0073] (a) Genomic DNA extraction: Genomic DNA was extracted using a genomic DNA extraction kit (Tiangen), and the concentration of genomic DNA (gDNA) was quantified using Nanodrop. The samples were stored at -20°C.

[0074] (b) Targeted Region PCR: Target regions were amplified from gDNA using a high-fidelity amplification enzyme (PrimeSTAR GXL DNA Polymerase, Takara). The primers are shown in Table 3 below. All deoxynucleotide sequences used were synthesized at the Shenzhen National Gene Bank Synthesis and Editing Platform. After observing a clear and single target band under a gel imaging system via 1% agarose gel electrophoresis (7.5V / em, 30min), the gel was excised and purified using a PCR purification and gel extraction kit (NucleoSpinExtract, MN). The concentration was measured using Nanodrop.

[0075] (c) Denaturation and Annealing: The mutant DNA and control group reaction system were mixed as shown in Table 5, and subjected to heat denaturation and annealing. The above program settings for the PCR instrument (Bio-rad) are shown in Table 6.

[0076] The transcription templates BEST12-AAVS1-gRNA2 were obtained as shown in SEQ ID NO: 15, and SpCas9-AAVS1-gRNA2 were obtained as shown in SEQ ID NO: 16.

[0077] (d) T7E1 digestion: Add 0.3 μL of T7EI nuclease to the reaction system in step (c), for a total of 20 μL. Perform the digestion reaction at 37℃ for 20 min.

[0078] (e) Activity detection: After the reaction is complete, add 4 μL of 6×Gel Loading Dye (NEB) and perform agarose gel electrophoresis to detect the bands (detailed steps are the same as the agarose gel electrophoresis operation described above).

[0079] Table 5 Annealing Reaction System

[0080] Component Amount DNA 200 ng 10X TE7I Buffer 2 μL ddH2O add to 19.7 μL

[0081] Table 6. Denaturation and Annealing Conditions

[0082] Temperature Time 95℃ 3 min -2°C / min 10 min 75℃ 30 sec -6°C / min 8 min 27℃

[0083] like Figure 2 As shown, the new BEST12 system has human cell editing activity, and it has genome editing activity at three sites of AAVS1 g1 / g2 / g5.

[0084] Example 4: Comparison of the in vitro catalytic activities of the new protein and SpCas9 at different temperatures

[0085] The following protocol was used to conduct in vitro cutting experiments using the gene editing system:

[0086] 1. Using the edited plasmid shown in Example 3 as the target, amplify to obtain the transcription template, and use MEGAshortscript. TM Kits are used for in vitro transcription of the corresponding gRNA. Primers and templates are shown in Table 7. Double-stranded DNA substrates are prepared using the primer pairs in Table 3, with DNA concentrations not less than 40 ng / μL and gRNA concentrations not less than 800 ng / μL.

[0087] 2. Mix 2 μL of 10×NEB buffer 3.1, 2 μL of 10-30 μM protein, and 1-2 μL of gRNA. Add RNase-free water to a final volume of 16 μL and vortex to mix. BEST12 and SpCas9 are... Figure 3 Incubate at the specified temperature for 15 minutes, add 4 μL of substrate DNA, and react at a gradient temperature for 1-5 hours.

[0088] 3. Take 10 μL of the product and perform electrophoresis on a 1% agarose gel.

[0089] Table 7. Primers and sequences for gRNA transcription template amplification.

[0090]

Claims

1. A Cas9 nuclease system comprising a Cas9 nuclease having the amino acid sequence shown in SEQ ID NO: 1 and gRNA, wherein the sequence of the gRNA scaffold is shown in SEQ ID NO:

3.

2. An isolated nucleic acid having a nucleotide sequence encoding the Cas9 nuclease of claim 1, and further comprising gRNA, or a DNA sequence corresponding to the gRNA.

3. The nucleic acid according to claim 2, wherein the nucleic acid is DNA or RNA.

4. An expression vector comprising the nucleic acid according to any one of claims 2-3.

5. A recombinant cell comprising the expression vector of claim 4.

6. A kit comprising the expression vector of claim 4 or the recombinant cells of claim 5.

7. Use of the Cas9 nuclease system of claim 1, the nucleic acid of any one of claims 2-3, the expression vector of claim 4, or the recombinant cell of claim 5 in gene editing.

8. The use according to claim 7, wherein the use is cell gene editing, nucleic acid detection, and high-throughput sequencing.

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