Protein capable of improving efficiency of Cas9 base editor and pilot editor and application

Abstract

The invention relates to the technical field of gene engineering, and discloses a protein capable of improving the efficiency of a Cas9 base editor and a pilot editor and application, the protein is obtained by cutting off N amino acids at the C terminal of a protein sequence from a fusion protein; the fusion protein is obtained by fusion of SpCas9 and Smac Cas9, and the Smac Cas9 is a fusion protein. After part of amino acid at the C end of the fusion protein is removed, the base editing efficiency and the pilot editing efficiency of the fusion protein are greatly improved. A base editor and a pilot editor are prepared from the protein, a new thought is provided for further improvement and development of a CRISPR / Cas gene editing technology, and a theoretical basis is provided for application of the gene editing technology in the fields of clinic, biotechnology and the like.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a protein and application thereof that can improve the efficiency of a Cas9 base editor and a lead editor. Background Art

[0002] Gene editing technologies such as base editing (BE) and prime editing (PE) based on the CRISPR / Cas system have shown great application value in gene editing, expression regulation, disease treatment, etc. Among them, Cas9 protein is a very critical component and is closely related to the problems of editing efficiency and off-target rate faced by current gene editing technologies.

[0003] In order to improve the efficiency of base editing, existing methods: for example, by fusing different deaminases and Cas proteins, base editors with higher editing efficiency can be constructed. For example, combining the efficient Cas12a variant hyperCas12a with the efficient single-stranded DNA deaminase, and then introducing the non-toxic mutant DddA-GSVG of the double-stranded DNA deaminase DddA, can significantly improve the base editing efficiency in rice; in addition, through artificial intelligence (AI) technology, such as convolutional neural networks (CNN), the editing efficiency of base editors can be predicted and optimized; multiple base editors can also be combined for multi-site editing. The optimization methods for improving the lead editor are currently mainly concentrated in the following aspects: optimization of RT reverse transcriptase, NLS sequence, modification of nCas9, and optimization of nCas9 nuclease activity. However, the current methods for improving the efficiency of base editing and lead editing are not very ideal. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a protein and application that can improve the efficiency of Cas9 base editors and lead editors.

[0005] The technical solution adopted by the present invention is:

[0006] A protein, wherein the protein is obtained by cutting off the C-terminal N amino acids of the protein sequence of a fusion protein; the fusion protein is obtained by fusing SpCas9 and SmacCas9.

[0007] Furthermore, the fusion protein is SpSmac Cas9-D3.

[0008] Furthermore, the SpSmac Cas9-D3 is obtained by fusion of the non-PID domain of SpCas9 and the PID domain of SmacCas9.

[0009] Furthermore, the N is 3.

[0010] Furthermore, the sequence of the fusion protein SpSmacCas9-D3 is SEQ ID NO.1; the protein sequence is SEQ ID NO.2.

[0011] A use of a protein for gene editing.

[0012] Furthermore, the protein is used to prepare Cas9 base editor or lead editor.

[0013] A protein-constructed base editor is constructed using ABE-NG and protein as templates.

[0014] A protein-constructed lead editor constructed using PE2 and protein as templates.

[0015] A method for improving the efficiency of Cas9 base editors and lead editors, using proteins to construct Cas9 base editors or lead editors for gene editing.

[0016] The beneficial effects of the present invention are:

[0017] The present invention greatly improves the efficiency of base editing and lead editing by removing some amino acids from the C-terminus of the fusion protein. The base editor and lead editor prepared by the protein of the present invention provide new ideas for the further improvement and development of CRISPR / Cas gene editing technology, and provide a theoretical basis for the application of gene editing technology in clinical and biotechnology fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram comparing the base editing efficiency of the proteins obtained in the examples of the present invention.

[0019] Figure 2 This is a schematic diagram comparing the lead editing efficiency of proteins obtained in the examples of the present invention. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0021] A protein obtained by truncating the C-terminal N amino acids of the protein sequence of the fusion protein;

[0022] The fusion protein is obtained by fusion of SpCas9 and Smac Cas9. The fusion protein is SpSmac Cas9-D3.

[0023] SpSmac Cas9-D3 is a fusion of the non-PID domain of SpCas9 and the PID domain of SmacCas9; wherein N is 3.

[0024] The sequence of the fusion protein SpSmacCas9-D3 is SEQ ID NO.1; specifically, it is as follows:

[0025] MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGA SQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRAMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEIQTVGQNGGLFDDNPKSPLEVTPSKLVPLKKELNPKKYGGYQKPTTAYPVLLITDTKQLIPISVMNKKQFEQNPVKFLRDRGYQQVGKNDFIKLPKYTLVDIGDGIKRLWASSKEIHKGNQLVVSKKSQILLYHAHHLDSDLSNDYLQNHNQQFDVLFNEIISFSKKCKLGKEHIQKIENVYSNKKNSASIEELAESFIKLLGFTQLGATSPFNFLGVKLNQKQYKGKKDYILPCTEGTLIRQSITGLYETRVDLSKIGED

[0026] The protein sequence is SEQ ID NO.2, as follows:

[0027]

[0028] Application of protein, protein is used in gene editing. Protein is used to prepare Cas9 base editor or lead editor. Base editor is constructed using ABE-NG and protein as template. Lead editor is constructed using PE2 and protein as template.

[0029] A method for improving the efficiency of Cas9 base editors and lead editors, using proteins to construct Cas9 base editors or lead editors for gene editing.

[0030] Example

[0031] Based on the existing SpSmacCas9, primers were designed and PCR amplification was performed to obtain a fragment with 3 amino acids at the C-terminus of the protein sequence truncated, which was named SpSmac-4 Cas9.

[0032] Primers were designed by snapgene; target fragments were amplified by PCR, and after the template was digested by Dpn1, gel recovery was performed for electrophoresis verification; linearized plasmids after site-directed mutagenesis were connected by seamless cloning; DH5α was used as the recipient cell for transformation, and plasmids were extracted after transformation; sequencing verification was performed by the Sanger method.

[0033] ABE and PE editors were constructed separately, endogenous sgRNA was constructed, and the editing effect of base editors was verified by endogenous targets. At the same time, pegRNA was constructed based on sgRNA to verify the editing efficiency of the lead editor.

[0034] The existing ABE-NG and PE2 prepared in our laboratory, as well as SpSmac Cas9 and SpSmac-4 Cas9 were used as templates and Vazyme high-fidelity polymerase was used to generate Max Super-Fidelity DNA Polymerase amplified the fragment, and the primers were synthesized by Qingke Biotechnology. The amplified DNA fragment was recovered by gel electrophoresis and then seamlessly cloned and constructed using the ClonExpressII One Step Cloning Kit. The editing efficiency of the obtained base editors at different target sites is shown in Figure 2. Figure 1 As shown in Figure 2, the editing efficiency of the lead editor at different target sites is as follows: Figure 2 shown.

[0035] 1. The reaction system and procedure of PCR amplification are shown in Tables 1, 2 and 3.

[0036] Table 1. Reaction system

[0037]

[0038] Table 2. Reaction procedures

[0039]

[0040] Table 3. Reaction system

[0041]

[0042] 2. The DNA gel recovery process is as follows:

[0043] i. After the PCR reaction is completed, all the PCR products to be recovered are loaded into the wells of the agarose gel to run out the target band.

[0044] ii. Use a clean scalpel or razor blade to cut a slice of the gel containing the DNA fragment. Cut as close to the DNA as possible to reduce the volume of the gel. Place the gel slice into a pre-weighed 1.5 mL centrifuge tube and weigh it. Record the weight of the gel slice.

[0045] iii. Add 1:1 volume of Binding Buffer (volume: weight) to the gel slices and incubate the gel mixture at 50-60°C for 10 minutes or until the gel slices are completely dissolved. Invert the centrifuge tube every few minutes to mix to promote the melting process. To ensure that the gel is completely dissolved, the gel mixture can be briefly vortexed before adding to the column.

[0046] iv. Transfer the gel solution dissolved in step iii to the DNA purification column (including the collection tube), adding up to 800 μL at a time. Centrifuge at 8000 rpm for 1 min, discard the liquid in the collection tube, and put the purification column back into the same collection tube.

[0047] v. Repeat the previous step until all the liquid has been added to the purification column.

[0048] vi. Add 700 μL of DNA wash buffer (diluted with anhydrous ethanol) to the DNA purification column. Centrifuge at 12,000 rpm for 1 min, discard the liquid in the collection tube, and put the purification column back into the same collection tube.

[0049] vii. Repeat step vi once, and then centrifuge the empty DNA purification column at 12000 rpm for 2 min to completely remove the residual washing buffer.

[0050] viii. Transfer the DNA purification column to a clean 1.5 mL centrifuge tube, open the lid, and evaporate the residual ethanol at room temperature or in a 55°C metal bath.

[0051] ix. Add an appropriate amount of 55°C incubated ddH2O to the center of the purification column membrane and centrifuge at 12000rpm for 2min. Remove the DNA purification column to obtain the purified PCR product.

[0052] 3. The reaction system and reaction conditions of homologous recombination are shown in Table 4

[0053] Table 4. Reaction system

[0054]

[0055] 4. Chemical transformation

[0056] i. Place competent cells in an ice bath (thaw for 1-2 minutes), add target DNA, mix gently, and place in an ice bath for 30 minutes. (The volume of DNA used should not exceed 1 / 10 of the volume of competent cell suspension)

[0057] ii. Place the centrifuge tube in a 42°C water bath for 90 seconds, then quickly transfer the centrifuge tube to an ice bath to cool the cells for 2 minutes. Do not shake the centrifuge tube during this process.

[0058] iii. Add 500 μL of sterile LB medium (without antibiotics) to each centrifuge tube, mix well, and culture in a shaker at 37°C and 220 rpm for 60 minutes to express the relevant resistance marker gene on the plasmid and revive the bacteria.

[0059] iv. Pipette an appropriate volume of transformed competent cells onto LB solid agar medium containing the corresponding antibiotics, and spread the competent cells evenly. Place the plate at room temperature until the liquid is absorbed, invert the plate, and culture at 37°C for 12-16 hours.

[0060] 5. The construction of the above sgRNA is as follows:

[0061] Construction of endogenous sgRNA:

[0062] Screen sgRNA near the pathogenic mutation site of the pathogenic gene and synthesize each sgRNA oligo dry powder

[0063] (1) spsgRNA scafford digestion

[0064] (2) sgRNA phosphorylation: Dissolve the oligo dry powder of each sgRNA into 100 μM and prepare the reaction as follows. Vortex to mix and react in a PCR instrument at 37°C for 30 min.

[0065] (3) Annealing: Place the product after the reaction into boiling water and slowly cool it down to room temperature.

[0066] (4) sgRNA ligation: Prepare the ligation reaction system as follows and react at 16°C for 2 hours.

[0067] (5) Transformation and single clone selection for verification.

[0068] (6) Expand the correct clone by shaking and culture, and extract the plasmid.

[0069] Endogenous pegRNA construction

[0070] (1) pegRNA primers

[0071] (2) PCR amplification

[0072] (3) Connection

[0073] (4) Transformation, single clone selection, and sequencing identification.

[0074] 6. The plasmid extraction process is as follows:

[0075] (1) Plasmid extraction

[0076] i. After ligation and transformation, the correct clone was inoculated into 5-10 mL of LB medium (with antibiotics added) and cultured at 37°C with shaking at 220 rpm for 12-16 h.

[0077] ii. Centrifuge at 12000 rpm for 2 min to collect the bacteria and remove the supernatant LB medium as much as possible.

[0078] iii. Add 250 μL Solution Ⅰ (with RNase A) to the collected bacteria and suspend them with a pipette or vortex to mix.

[0079] iv. Add 250 μL of Solution II to the liquid in the previous step, gently invert and mix about 10 times, and let it stand for two minutes.

[0080] v. Add 350 μL of Solution III to the liquid in the previous step, immediately mix by inverting until white flocculent precipitate appears, and then centrifuge at 12,000 rpm for 10 minutes.

[0081] vi. Add the supernatant after centrifugation to the adsorption column with a collection tube, centrifuge at 8000rpm for 1 minute, add the liquid in the collection tube back to the adsorption column and centrifuge once. Then discard the liquid in the collection tube.

[0082] vii. Add 500 μL HBC Buffer to the adsorption column, centrifuge at 12,000 rpm for 1 minute, and then discard the liquid in the collection tube.

[0083] viii. Add 500 μL DNA Wash Buffer to the adsorption column, centrifuge at 12000 rpm for 1 minute, and then discard the liquid in the collection tube. Repeat this step once.

[0084] ix. Centrifuge the empty adsorption column at 12000 rpm for 2 min to completely remove the residual DNA Wash Buffer.

[0085] x. Transfer the adsorption column to a clean 1.5 mL centrifuge tube, open the lid, and evaporate the residual ethanol at room temperature or in a 55°C metal bath.

[0086] xi. Add an appropriate amount of 55℃ incubated ddH2O to the center of the adsorption column membrane and centrifuge at 12000rpm for 2min. Remove the adsorption column to obtain the plasmid. Quantify using NanoDROP2000 and store in a -20℃ refrigerator for later use.

[0087] (2) Endotoxin-free plasmid extraction

[0088] i-iv. Same plasmid mini-prep steps i-iv

[0089] v. Add 125 μL N3 Buffer to the liquid in the previous step, mix by inverting 10 times until a white flocculent precipitate appears. Centrifuge at 12,000 rpm for 10 minutes.

[0090] vi. Transfer the supernatant after centrifugation to a clean 1.5 mL centrifuge tube, add 0.1 volume of ETR Buffer, and mix by inverting several times. The liquid will become turbid.

[0091] vii. Place the centrifuge tube on ice and incubate for 10 minutes. During this time, invert the centrifuge tube several times. The liquid will become clear. Then place it in a 42°C water bath for 5 minutes. The solution will become turbid again.

[0092] viii. Centrifuge the liquid from the previous step at 12,000 rpm for 3 minutes, then transfer the supernatant to a new 1.5 mL centrifuge tube, add 0.5 times the volume of anhydrous ethanol, mix by inverting, and incubate for 3 minutes.

[0093] ix. Steps vi-ix of plasmid mini-prep.

[0094] 7. Cell culture process

[0095] The cells were cultured in DMEM medium containing 10% fetal bovine serum (Excell) and penicillin-streptomycin. The incubator was maintained at 37°C and 5% CO2. The cells were subcultured when the cell density reached 90%.

[0096] (1) Cell recovery

[0097] ①DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin solution) was placed in

[0098] Preheat in a 37℃ constant temperature water bath for 15 minutes and set aside;

[0099] ②Take out the cryopreservation tube from the liquid nitrogen tank, immediately place it in a 37°C constant temperature water bath and gently shake it to freeze.

[0100] tube until the medium is completely melted;

[0101] 13 Hangzhou Normal University Master's Thesis 2 Analysis of PID Domain Exchange Recombination of Natural Cas9 Family Genes

[0102] ③ Transfer the cell suspension into a 15 mL centrifuge tube, slowly add 5 mL of complete medium, and centrifuge at 8000 rpm for 5 min;

[0103] ④ Remove the supernatant medium, suspend the precipitated cells with new medium, and divide the cell fluid into

[0104] Add to culture dishes of different sizes;

[0105] ⑤After marking the recovery date and cell generation number, place the culture dish in a cell culture incubator for culture.

[0106] (2) Cell passaging

[0107] ① DMEM complete medium, 1×PBS and trypsin digestion solution (containing EDTA) were kept at 37°C

[0108] Preheat in a water bath for 15 minutes and set aside;

[0109] ② Aspirate the culture medium, add 2 mL PBS to wash, and aspirate the PBS;

[0110] ③ Add 1 mL of trypsin digestion solution and shake gently to ensure that the cells are fully digested;

[0111] ④ When cells are observed to fall off the bottom of the culture dish with the naked eye and the trypsin solution becomes turbid, add 3 mL of DMEM

[0112] Complete medium was pipetted to mix well to terminate digestion;

[0113] ⑤ Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 800 rpm for 5 minutes;

[0114] ⑥ Aspirate and discard the supernatant medium, add 1 mL of fresh medium, and mix by pipetting;

[0115] ⑦According to the experimental requirements, take an appropriate amount of cell suspension and add it to the culture dish prepared in advance;

[0116] ⑧Gently shake the tube and place it in the incubator after observing it under a microscope.

[0117] (3) Cell cryopreservation

[0118] ① Preheat DMEM complete medium, 1× PBS and trypsin digestion solution (containing EDTA) in a 37℃ constant temperature water bath for 15 minutes and set aside;

[0119] ② Aspirate the culture medium, add 2 mL PBS to wash, and aspirate the PBS;

[0120] ③ Add 1 mL of trypsin digestion solution and shake gently to ensure that the cells are fully digested;

[0121] ④ When cells are observed to fall off the bottom of the culture dish with the naked eye and the trypsin solution becomes turbid, add 3 mL of DMEM

[0122] Complete medium was pipetted to mix well to terminate digestion;

[0123] ⑤ Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 8000 rpm for 5 min;

[0124] ⑥ Aspirate and discard the supernatant culture medium, add 1 mL of pre-cooled cell freezing solution, which is composed of 9:1 complete culture medium and DMSO, and mix by pipetting;

[0125] ⑦ Transfer the cells into cryopreservation tubes, seal the tubes with sealing film, and mark the cell types, passage times and

[0126] Date of freezing;

[0127] ⑧Place the cryovials at 4°C for 20 min, -20°C for 1 h, -80°C overnight and then transfer to liquid nitrogen.

[0128] Before the above cell experiments are performed, the cell laboratory and the biosafety cabinet must be disinfected with ultraviolet light.

[0129] All items brought into the clean bench must be disinfected with 75% alcohol for more than 30 minutes.

[0130] (4) Cell transfection process

[0131] 1) One day before transfection, seed an appropriate amount of cells to achieve 80-90% of the cell density for transfection the next day, such as 1-4×10 4 .

[0132] 2) Prepare DNA-Buffer Transeasy complex by mixing as follows

[0133] i. Dilute the appropriate amount of DNA in 25 μL Opti-MEM serum-free medium and mix well. (For base editing experiments, the amount of transfected DNA is 300 ng editor plasmid, 100 ng sgRNA plasmid and 30 ng puromycin selection plasmid. For experiments to determine PAM, the amount of transfected DNA is 300 ng editor plasmid, 100 ng sgRNA plasmid and 45 ng PAM plasmid library.)

[0134] ii. Gently mix BufferTranseasy, pipette 0.75 μL volume and dilute it in 25 μL Opti-MEM serum-free medium, mix well and place at room temperature for 5 minutes.

[0135] iii. After 5 minutes, mix the diluted DNA and BufferTranseasy, a total of 50 μL, mix well and leave at room temperature for 20 minutes to form a complex.

[0136] iv. Add 50 μL DNA-BufferTranseasy complex to the cell culture dish and gently shake the dish to mix.

[0137] v. 24 hours after transfection, the culture medium was replaced with fresh complete medium containing 3 μg / mL puromycin, and the cell genome was collected after further culture for 48 hours.

[0138] (5) Cell genome extraction process

[0139] 1) After transfection, remove the culture medium from the cells and add 50 μL 1× PBS to each well. Gently shake to wash away excess culture medium and dead cells. Then remove and discard the PBS. Repeat the washing process three times.

[0140] 2) Add 100 μL 1× PBS to each well, blow down the cells with a pipette, put them into a centrifuge tube, and centrifuge at 12,000 rpm for 3 minutes at room temperature.

[0141] 3) Discard the supernatant, add 30 μL of cell lysis working solution to the cell pellet, blow evenly with a pipette, then incubate in a 55°C metal bath for 20 minutes and inactivate at 95°C for 10 minutes. The cell genome is stored in a -20°C refrigerator for subsequent experiments.

[0142] Using the extracted genome as a template, corresponding genome amplification primers were designed near each target site, PCR amplification was performed on the target site, and the amplified product was subjected to Sanger sequencing. The sequencing results were input into the EditR software to analyze the editing efficiency.

[0143] from Figure 1 and Figure 2It can be seen that compared with the editing results of the fusion protein without truncation of amino acids, the base editing and lead editing efficiencies were significantly improved after deleting the three amino acids at the C-terminus of SpSmacCas9. Figure 1 Sp-Smac is the base editor result obtained by using fusion protein, and Sp-Smac-4 is the base editor result obtained by deleting the three amino acids at the C-terminus. The three groups are the base editing efficiencies obtained by testing different targets.

[0144] Figure 2 In the figure, 46-GAAA+6A-G, S10-AAAA+6A-C, S3-GAAG+6A-C, S4-TAAG+6A-C, and S10-AAAA+6A-C are the selected target positions. The results on the left are the test results of the lead editor constructed by deleting the three amino acids at the C-terminus, and the results on the right are the test results of the lead editor constructed using the fusion protein SpSmacCas9.

[0145] After the amino acids at the C-terminus of the fusion protein are removed in the present invention, the base editing and lead editing efficiencies are significantly improved.

Claims

1. A protein, characterized in that The protein is obtained by cutting off the C-terminal N amino acids of the protein sequence of the fusion protein; The fusion protein is obtained by fusion of SpCas9 and SmacCas9.

2. A protein according to claim 1, characterized in that The fusion protein is SpSmac Cas9-D3.

3. A protein according to claim 2, characterized in that The SpSmac Cas9-D3 is obtained by fusing the non-PID domain of SpCas9 and the PID domain of SmacCas9.

4. A protein according to claim 3, characterized in that The N is 3.

5. A protein according to claim 3, characterized in that The sequence of the fusion protein SpSmac Cas9-D3 is SEQ ID NO.1; the protein sequence is SEQ ID NO.

2.

6. The use of any protein according to claim 1 to 5, characterized in that: The protein is used in gene editing.

7. The use according to claim 6, characterized in that: The protein is used to prepare Cas9 base editors or lead editors.

8. A base editor constructed using any one of the proteins of claims 1 to 5, characterized in that: It was constructed using ABE-NG and protein as templates.

9. A lead editor constructed using any one of the proteins of claims 1 to 5, characterized in that: It was constructed using PE2 and protein as templates.

10. A method for improving the efficiency of Cas9 base editors and lead editors, characterized in that: Use protein-constructed Cas9 base editors or lead editors for gene editing; The protein is any one of claims 1 to 5.

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