Research and development and application of microbe-derived RecE protein mediated human-derived cell long-fragment precise gene editing system

Abstract

The invention belongs to the technical field of biological medicine. A series of novel gene editing systems spCas9-RecE, spCas9 (D10A)-RecE, spCas9 (H840A)-RecE and dCas9-RecE are created through humanized application of microorganism-derived RecE protein, the HDR-mediated precise gene editing efficiency in the gene editing process is improved, the safety risk caused by DNA fracture damage accompanying in the traditional gene editing process is remarkably reduced, and the gene editing efficiency is improved. Potential safety hazards of gene editing in gene therapy and cell therapy transformation application processes are eliminated, and a new tool is provided for development of precision medical treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the development and application of a long-fragment precise gene editing system for human cells mediated by microbial-derived RecE protein. Background Art

[0002] As a new synthetic biology system, the CRISPR / Cas9 genome editing tool provides powerful technical support for genetic engineering, driving the development of basic scientific research and the healthcare industry. In particular, for major diseases such as gene mutation-related diseases and tumors, CRISPR / Cas9-based cell therapy strategies are constantly emerging, bringing good news to patients. In 2023, the UK Medicines and Healthcare Products Regulatory Agency (MHRA) and the US Food and Drug Administration (FDA) successively approved the world's first CRISPR / Cas9 cell therapy product, Casgevy, for the treatment of the inherited blood disorder sickle cell disease, marking the beginning of gene therapy 2.0.

[0003] However, existing gene editing systems have many limiting factors in the process of clinical gene therapy translation and application. For example, existing gene therapy strategies mostly focus on knocking out pathogenic mutant genes, rather than precisely repairing long DNA fragments or inserting them into the genome. Existing systems all require the spCas9 nuclease to introduce double-strand breaks in DNA at specific sites in the genome. The broken DNA will be cytotoxic to the targeted cells and induce apoptosis. The off-target effects of the Cas9 nuclease bring safety risks to its application. Single-cell sequencing results show that existing methods have a certain probability of inducing chromosome isomerism, recombination, and fragment loss in edited cells.

[0004] Therefore, the research and development of safe, accurate and efficient long-fragment (kilobase level) gene editing tools remains one of the key factors in promoting the further development of clinical gene therapy translational applications. Summary of the Invention

[0005] This patent successfully created a series of gene editing systems by humanizing the microbial-derived protein RecE, significantly improving the efficiency of long-fragment editing of eukaryotic genomes mediated by spCas9, spCas9(D10A), spCas9(H840A) and dCas9. In particular, the spCas9(D10A)-RecE, spCas9(H840A)-RecE and dCas9-RecE gene editing systems achieve long-fragment precise gene editing operations without generating DNA double-strand breaks, breaking through the safety risks of traditional gene editing tools and providing new tools for the development of in vivo and in vitro gene therapy and cell therapy.

[0006] The purpose of the present invention is to provide a series of novel long-fragment gene editing systems that can achieve precise, safe, and efficient long-fragment gene editing operations in eukaryotic genomes, especially avoiding safety hazards such as DNA double-strand breaks associated with the gene editing process, and confirming their gene editing capabilities at the cell line and primary iPSC cell levels, providing effective application tools for in vivo and in vitro gene therapy and cell therapy of gene mutation-related diseases, and promoting the development of clinical treatment.

[0007] The technical solutions of the present invention are as follows:

[0008] The first objective of the present invention is to provide the use of a microbial protein, RecE, in constructing a gene editing system. The microbial protein, RecE, can enhance the efficiency of long-range, precise gene editing in human cells mediated by the CRISPR / Cas9 gene editing system in eukaryotic cells. The full name of the RecE protein described herein is EcR RecE, and the nucleotide sequence of the RecE protein is shown in SEQ ID NO. 17.

[0009] The second object of the present invention is to provide a CRISPR / Cas9 gene editing system based on the microbial protein RecE (spCas9-RecE), which consists of the following three elements: an MS2-sgRNA-Cas9 gene editing system expression vector, an MCP-RecE expression vector and an HDR homologous repair template.

[0010] Furthermore, the MCP-RecE expression vector includes an MCP-RecE sequence, i.e., the microbial protein RecE and the MCP aptamer protein are fused and expressed;

[0011] Preferably, the base vector of the MCP-RecE expression vector is the EF1A-MCP expression vector, and the RecE sequence is inserted between the BamHI and EcoRI restriction sites of the EF1A-MCP expression vector to construct the vector.

[0012] The base vector of the MCP-RecE expression vector is the EF1A-MCP expression vector, and its sequence is shown in SEQ ID NO.15.

[0013] Furthermore, the gene sequence of the MCP-RecE expression vector is shown as SEQ ID NO.9, the nucleotide sequence of the microbial protein RecE is shown as SEQ ID NO.17, and the amino acid sequence of MCP-RecE is shown as SEQ ID NO.10.

[0014] Furthermore, the MS2-sgRNA-Cas9 gene editing system expression vector includes the key components for gene editing, the MS2-sgRNA sequence, and the Cas9 nuclease. The MS2-sgRNA sequence includes the MS2-sgRNA scaffold sequence, and the Cas9 nuclease is selected from spCas9 or its related variant sequence.

[0015] In addition, the expression vector may also contain a T2A-EBFP sequence for evaluating cell transfection efficiency.

[0016] In a particular embodiment, the base vector of the MS2-sgRNA-Cas9 gene editing system expression vector is the pU6-CBh-T2A-EBFP expression vector having a sequence as shown in SEQ ID NO. 16. The base vector already includes the T2A-EBFP sequence.

[0017] In the above situation, the MS2-sgRNA scaffold sequence was ligated between the NdeⅠ restriction site of the pU6-CBh-T2A-EBFP expression vector, and the Cas9 nuclease sequence was ligated between the AgeⅠ and HindⅢ restriction sites.

[0018] Furthermore, the spCas9-related variant in the MS2-sgRNA-Cas9 gene editing system expression vector is selected from spCas9 (D10A), spCas9 (H840A) or dCas9.

[0019] Furthermore, when the Cas9 nuclease in the MS2-sgRNA-Cas9 gene editing system expression vector is spCas9, the gene sequence of the MS2-sgRNA-spCas9 gene editing system expression vector is shown in SEQ ID NO.1; when the Cas9 nuclease in the MS2-sgRNA-Cas9 gene editing system expression vector is the spCas9 variant dCas9, the gene sequence of the MS2-sgRNA-dCas9 gene editing system expression vector is shown in SEQ ID NO.2.

[0020] The MS2-sgRNA-Cas9 gene editing system expression vector also includes a spacer sequence targeting the gene site to be edited; the spacer sequence targeting the gene site to be edited is connected between the BbsⅠ restriction sites of the MS2-sgRNA scaffold sequence in the MS2-sgRNA-Cas9 gene editing system expression vector shown in SEQ ID NO.1 or SEQ ID NO.2; so that the MS2-sgRNA scaffold sequence and the spacer sequence together constitute the MS2-sgRNA sequence.

[0021] Preferably, the gene to be edited is selected from the group consisting of ACTB human gene, HSP90AA1 human gene, DYNLT1 human gene, EMX1 human gene, VEGFA human gene, and AAVS1 human gene;

[0022] Preferably, the spcaer sequence of the ACTB human gene is specifically shown as SEQ ID NO.3, the spcaer sequence of the HSP90AA1 human gene is specifically shown as SEQ ID NO.4, the spcaer sequence of the DYNLT1 human gene is specifically shown as SEQ ID NO.5, the spcaer sequence of the EMX1 human gene is specifically shown as SEQ ID NO.6, the spcaer sequence of the VEGFA human gene is specifically shown as SEQ ID NO.7, and the spcaer sequence of the AAVS1 human gene is specifically shown as SEQ ID NO.8.

[0023] Based on the working principle of the SAM recruitment system, the MCP adaptor of the MCP-RecE expression vector specifically binds to the MS2 stem-loop structure of the MS2-sgRNA-Cas9 gene editing system expression vector, enabling RecE and sgRNA-Cas9 to synergistically exert gene editing functions in eukaryotic cells.

[0024] The HDR homology repair template includes a P2A-mKate fluorescent protein expression sequence of approximately 1 kb and a homology arm DNA sequence of the gene to be edited located on the left and right of the P2A-mKate fluorescent protein expression sequence, wherein the gene to be edited is selected from the human ACTB gene, the human HSP90AA1 gene, the human DYNLT1 gene, the human EMX1 gene, the human VEGFA gene or the human AAVS1 gene.

[0025] Furthermore, except for the DTNLT1 human gene, the DNA sequences of the left and right homologous arms of the other genes to be edited are both 600 bp or longer, and the DNA sequences of the left and right homologous arms of the DYNLT1 human gene are 200 bp and 400 bp or longer, respectively;

[0026] Furthermore, the P2A-mKate fluorescent protein expression sequence of the HDR homology repair template is shown in SEQ ID NO.11;

[0027] Furthermore, the HDR homologous repair template sequence of the ACTB human gene is shown as SEQ ID NO.12, the HDR homologous repair template sequence of the HSP90AA1 human gene is shown as SEQ ID NO.13, the HDR homologous repair template sequence of the DYNLT1 human gene is shown as SEQ ID NO.14, the HDR homologous repair template sequence of the EMX1 human gene is shown as SEQ ID NO.18, the HDR homologous repair template sequence of the VEGFA human gene is shown as SEQ ID NO.19, and the HDR homologous repair template sequence of the AAVS1 human gene is shown as SEQ ID NO.20.

[0028] The third object of the present invention is to provide the use of the aforementioned gene editing system in the preparation of drugs or reagents based on cell therapy and / or gene therapy related diseases.

[0029] Furthermore, the cell therapy is based on HEK293T cells, A549 cells, HepG2 cells or Hela cell lines.

[0030] The application can improve the efficiency of long-fragment gene editing at multiple sites in cells;

[0031] Preferably, the disease is autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive hepatolenticular degeneration, sickle cell disease or β-thalassemia, Duchenne muscular dystrophy, congenital amaurosis, cervical cancer, etc. The precise, safe, and efficient gene editing mediated by the spCas9-RecE gene editing system of the present invention is mainly achieved by the following steps:

[0032] Cell culture:

[0033] HEK293T and other human cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) high glucose medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / ml streptomycin in a 5% CO 2 , 37° C. constant temperature cell culture incubator.

[0034] Cell Thawing: Remove the cells to be revived from liquid nitrogen and quickly dissolve them in 37°C warm water. Once completely dissolved, pipette the cryopreservation solution into a 15ml centrifuge tube and centrifuge at 200xg for 3 minutes. After centrifugation, discard the supernatant and resuspend the cells in 1ml of culture medium. Add 1ml of cell suspension to a 10cm cell culture dish containing 9ml of DMEM complete medium (supplemented with 10% fetal bovine serum, 100U / ml penicillin, and 100μg / ml streptomycin). Incubate in a humidified, 5% CO2, 37°C incubator. Change the medium after the cells attach.

[0035] Cell passaging: When cells reach 80-90% confluence, discard the supernatant and wash once with DPBS. Add 0.05% Trypsin-EDTA and incubate at 37°C for 1 min. When cells become spherical and slightly detached, add DMEM supplemented with 10% fetal bovine serum to terminate the digestion. Transfer the cell suspension to a 15 ml centrifuge tube and centrifuge at 200 x g for 3 min. After centrifugation, discard the supernatant and resuspend the cells in 1 ml of complete DMEM. Passage the cells at the desired ratio.

[0036] Cell cryopreservation: When cell confluence reaches 95%, discard the supernatant, wash once with DPBS, digest the cells with 0.05% Trypsin-EDTA, transfer to a 15ml centrifuge tube, and centrifuge at 200xg for 3 minutes. After centrifugation, discard the supernatant and resuspend the cells in the prepared cell freezing solution (DMEM:FBS:DMSO = 7:2:1). Transfer the cell freezing solution containing HEK293T cells to a 2ml cryovial, freeze at -80°C overnight, and then transfer to liquid nitrogen for storage.

[0037] Cell transfection:

[0038] 14-16 h before transfection, HEK293T cells were cultured at a density of 7.0-8.0 × 10 4 Cells were plated in a 48-well plate at a density of 100 cells / well and transfected when the cell confluence reached 60-70%. TM Dilute P3000 TM and Lipofectamine 3000 TM Each 48-well well was treated with 12.5 μl Opti-MEM TM / P3000 TM Dilute 500 ng of plasmid DNA with the premix and then add an equal volume of Opti-MEM TM / Lipofectamine 3000 TM The premix was allowed to stand for 15 minutes. 25 μl of the mixed transfection reagent was added to each well of cells for transfection. The 48-well plate was gently shaken to make it evenly distributed in the culture medium. 48 hours after transfection, the cell culture medium was replaced. 72 hours after transfection, the cells were collected for subsequent corresponding experimental tests. Hela and HepG2 cells were cultured at 3.0×10 4 Cells were plated at a density of 500 cells / well in a 48-well plate. Each well was transfected with 500 ng of DNA plasmid. Cell culture medium was changed 6 and 48 hours after transfection. Cells were harvested 72 hours after transfection for subsequent assays.

[0039] Streaming analysis:

[0040] The efficiency of mKate fluorescent protein insertion was quantified by flow cytometry. 72 hours after transfection, the supernatant was discarded, the cells were washed once with DPBS, and digested with 0.05% Trypsin-EDTA. Each well of cells was transferred to a 1.5 ml EP tube and centrifuged at 200 x g for 3 minutes. After centrifugation, the supernatant was discarded and the cells were resuspended in DPBS supplemented with 2% fetal bovine serum. Gene editing efficiency was analyzed by flow cytometry within 2 hours.

[0041] The key components of the pCas9-RecE gene editing system, MCP-ReE and spCas9-MS2-sgRNA or its related variant expression vectors, are mainly achieved through the following steps:

[0042] Plasmid construction:

[0043] The gene sequences of spCas9-related variants such as EcR RecE, spCas9-sgRNA-MS2, or dCas9 were obtained by whole gene synthesis (in certain specific embodiments, the sequence information is shown in SEQ ID NO. 1 / 2 / 9), and EcRRecE was ligated into the EF1A-MCP expression vector plasmid, or sRNA-MS2-spCas9 or its related variants were ligated into the hU6-CBh-T2A-EBFP expression vector plasmid by Gibson test. After the Gibson test, the products were placed on ice for E. coli competent transformation experiments.

[0044] Plasmid transformation:

[0045] Thaw the competent E. coli cells on ice. Once completely thawed, add the plasmid to be transformed to the competent E. coli cells at a 1:10 volume ratio. Gently stir with a pipette to mix thoroughly. Let stand on ice for 10 minutes. Streak the competent mixture onto LB solid medium containing ampicillin and incubate at 37°C for 12-18 hours.

[0046] Plasmid extraction:

[0047] After a single colony has grown on LB solid medium, pick a single colony and add it to LB liquid medium containing ampicillin. Incubate at 37°C in an air shaker for 12-14 hours. Collect the bacterial liquid and extract the plasmid. The specific experimental steps are as follows:

[0048] 1. Take 4 ml of bacterial suspension and centrifuge at 12,000 rpm for 30 seconds. Discard the supernatant and collect the bacterial pellet.

[0049] 2. Add 250 μl of solution P1 to resuspend the bacterial pellet and vortex until completely suspended.

[0050] 3. Add 250 μl of solution P2, gently invert the tube up and down 6 times to fully lyse the bacteria, and let it stand at room temperature for 4 minutes.

[0051] 4. Add 350 μl of solution P3 and immediately and gently invert the tube upside down 6 times; after a white flocculent precipitate appears, centrifuge at 12,000 rpm for 10 minutes.

[0052] 5. After centrifugation, carefully transfer the supernatant to the adsorption column (the adsorption column needs to be activated), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube.

[0053] 6. Add 500 μl of deproteinized solution PR, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube.

[0054] 7. Add 600 μl of WB rinse buffer and centrifuge at 12,000 rpm for 30 seconds. Discard the waste liquid in the collection tube. Repeat this process once more. Discard the waste liquid and return it to the collection tube. Centrifuge at 15,000 rpm for 3 minutes to remove as much of the rinse buffer as possible.

[0055] 8. Remove the adsorption column and transfer it to a 1.5ml EP tube. Uncap and let it stand for 3-5 minutes. Add 40μl of elution buffer EB to the center of the adsorption membrane. Let it stand at room temperature for 10 minutes. Centrifuge at 15,000 rpm for 3 minutes. Determine the concentration of the extracted plasmid.

[0056] Plasmid sequencing:

[0057] The extracted plasmid was subjected to Sanger sequencing at a concentration of 50 ng / μl, and the sequencing results were compared to ensure that the inserted fragment was correct. The plasmid with the correct final insertion result was selected for cell liposome transfection experiment.

[0058] The beneficial effects of the present invention are

[0059] Compared with traditional long-fragment gene editing systems, the novel gene editing system RecE provided by the present invention has the following advantages:

[0060] 1) The efficiency of long-range gene editing has been significantly improved, especially when applied to the dCas9 system;

[0061] 2) Effectively avoid the safety risks associated with DNA double-strand breaks during traditional gene editing;

[0062] 3) Stable in different cell lines, different genome editing sites, and different types of gene editing systems;

[0063] 4) The off-target risk in gene editing has been significantly improved.

[0064] This type of gene editing system provides a safe, accurate and efficient gene editing tool for clinical gene therapy and cell therapy, helping to promote the rapid development of precision medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 RecE eukaryotic expression vector;

[0066] (A) This vector is an MS2-sgRNA-spCas9 expression vector, containing the MS2-sgRNA and spCas9 sequences, as well as the CBh and human U6 promoter sequences, which are used to drive the expression of spCas9 and MS2-sgRNA, respectively. In addition, the vector also contains the EBFP fluorescent protein expression sequence, T2A-EBFP, and the EBFP fluorescent protein positive rate reflects the cell transfection efficiency.

[0067] (B) This vector is an MCP-RecE expression vector, which includes the EF1A promoter sequence and the MCP-RecE sequence.

[0068] Figure 2 HDR restoration template schematic and sequence;

[0069] (A) The HDR repair template is approximately 2 kb and contains approximately 1 kb of the P2A-mKate fluorescent protein expression sequence and the left and right homology arms of the ACTB, HSP90AA1, DYNLT1, EMX1, VEGFA, and AAVS1 human genes;

[0070] (B) Schematic diagram of the HDR repair template sequence of the HSP90AA1 human gene shown in SEQ ID NO. 13, wherein the bold sequence is the P2A-mKate fluorescent protein expression sequence, and the sequences before and after the bold sequence are the homology arm DNA sequences.

[0071] Figure 3 The spCas9-RecE gene editing system can effectively improve the efficiency and accuracy of precise gene editing in HEK293T cells. EcR RecE can effectively mediate the precise insertion of approximately 1kb exogenous P2A-mKate sequences into the human gene loci of HSP90AA1 (A), DYNLT1 (B), and AAV1 (C) in the HEK293T cell genome.

[0072] (D) Fluorescence image of EcR RecE-mediated insertion of P2A-mKate into the HSP90AA1 human gene locus; Cas9 only: control group without EcR RecE. Figure 4The spCas9-RecE gene editing system can effectively improve the efficiency of long-fragment gene editing in multiple eukaryotic cell lines; the spCas9-RecE gene editing system can mediate the precise insertion of approximately 1kb exogenous fragments at multiple sites in A549 (A), HepG2 (B) and Hela (C) cell lines, with knock-in efficiency significantly higher than that of the control group; the spCas9-RecE gene editing system can achieve a knock-in efficiency of 15-20% in Hela cell lines; NC: no sgRNA control group; Cas9 only: no EcR RecE control group.

[0073] Figure 5 The spCas9-RecE gene editing system can effectively reduce safety risks such as off-target effects;

[0074] Primers were designed to identify common off-target sites of spCas9 at the human EMX1 (A) and VEGFA (B) loci for NGS sequencing. The results showed that overexpression of EcR RecE did not increase the off-target efficiency of spCas9.

[0075] (C) Guide-seq structures of the EMX1 and VEGFA human gene loci showed that the genome-wide off-target efficiency of the EcR RecE group was similar to or lower than that of the control group;

[0076] (D) Statistical graph of off-target editing in different chromosomes of the genome in the control group and the EcR RecE group.

[0077] Figure 6 The spCas9(D10A) / spCas9(H840A) / dCas9-RecE gene editing system can effectively improve the efficiency of long-fragment gene editing at multiple sites in HEK293 cells;

[0078] (A) EcR RecE was applied to spCas9(D10A) and spCas9(H840A). The results showed that the editing efficiency of the RecE group was significantly higher than that of the control group and comparable to that of the published gene-editing tool SSAP / RecT (see article: Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects, 2021). NC: no sgRNA control; SSAP / RecT: gene-editing positive control.

[0079] (B) Using RecE in the dCas9 system, the results show that the editing efficiency of the RecE group is comparable to that of the positive control dCas9-SSAP. NC: no sgRNA control; SSAP / RecT: dCas9-RecT positive control. DETAILED DESCRIPTION

[0080] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0081] Example 1 Construction of expression vector for spCas9-RecE eukaryotic gene editing system

[0082] This project uses the SAM recruitment system to construct the RecE eukaryotic expression vector ( Figure 1 ).

[0083] 1. Figure 1 The construction process of the expression plasmid vector of MS2-sgRNA-spCas9 or its related variants shown in A is as follows:

[0084] (1) The gene sequences of MS2-sgRNA scaffold, spCas9 or their related variants were obtained by whole gene synthesis.

[0085] (2) Construction of the basic plasmid of the MS2-sgRNA-spCas9 gene editing system expression vector. The pU6-CBh-T2A-EBFP expression vector (the sequence of the pU6-CBh-T2A-EBFP expression vector is shown in SEQ ID NO. 16) was digested with restriction endonucleases NdeⅠ (FD0583, ThermoFisher), AgeⅠ (FD1464, ThermoFisher), and HindⅢ (FD0504, ThermoFisher) and incubated at 37°C in a water bath for 40 min. The digestion products were identified by 1.5% agarose gel electrophoresis and recovered using a Gel and PCR Clean-up Kit (Promega).

[0086] (3) Construction of the MS2-sgRNA-spCas9 gene editing system expression vector. The MS2-sgRNA scaffold and spCas9 gene sequences were connected to the vector plasmid by Gibson test with the enzyme digestion product of step (2) at a molar ratio of 1:2. The MS2-sgRNA scaffold sequence was connected between the NdeⅠ restriction site, and the spCas9 sequence was connected between the AgeⅠ and HindⅢ restriction sites. T5 nuclease (2×MultiF Seamless Assembly Mix, Abclonal) was mixed with the product at a volume of 1:1. The Gibson ligation reaction volume was 6 μl and the reaction was carried out at 50°C for 15 minutes. The MS2-sgRNA-spCas9 gene editing system expression vector was constructed.

[0087] (4) Transformation of competent cells: Thaw the competent E. coli on ice. After the competent cells are completely thawed, add the Gibson ligation product obtained in step (3) to the competent E. coli cells at a volume ratio of 1:10. Mix gently with a pipette tip and let stand on ice for 10 minutes. Streak the competent cell mixture onto LB solid medium containing ampicillin and incubate at 37°C for 12-18 hours.

[0088] (5) Monoclonal colony expansion and plasmid extraction: After a monoclonal colony grows on LB solid medium, pick a single colony and add it to LB liquid medium containing ampicillin. Incubate at 37°C in an air shaker for 12-14 h. Collect 4 ml of bacterial suspension and extract the plasmid using a high-purity plasmid extraction kit (Y2204, TIANGEN). The plasmid concentration is measured using a Multiskan SkyHigh full-wavelength microplate reader (A51119700DPC, ThermoScientific).

[0089] (6) Plasmid sequencing: The extracted plasmid was sent to a sequencing company at a concentration of 50 ng / μl for Sanger sequencing. The sequencing results were compared to ensure that the inserted fragments were correct. The plasmid with the correct final insertion result was selected for cell transfection experiments. The gene sequence of the MS2-sgRNA-spCas9 gene editing system expression vector is shown in SEQ ID NO.1.

[0090] (7) Related variants of spCas9 include two nickases, spCas9 (D10A), spCas9 (H840A), and dCas9 with damaged nuclease activity. The construction of these overexpression vector plasmids is based on the MS2-sgRNA-spCas9 gene editing system expression vector. The spCas9 gene sequence is removed using restriction endonucleases AgeⅠ (FD1464, ThermoFisher) and HindⅢ (FD0504, ThermoFisher) and the dCas9 and other spCas9 related variant gene sequences are connected into the vector by Gibson test. The gene sequence of the MS2-sgRNA-dCas9 gene editing system expression vector is shown in SEQ ID NO.2.

[0091] (8) In order to enable the constructed MS2-sgRNA-spCas9 or its related variant gene editing system expression vector plasmid to effectively target different human gene sites such as ACTB, HSP90AA1, DYNLT1, EMX1, VEGFA and AAVS1 for gene editing in various human cell lines such as HEK293T, a 20 bp spacer sequence targeting different gene sites was inserted into the BbsⅠ restriction endonuclease site of the MS2-sgRNA scaffold sequence in the MS2-sgRNA-spCas9 or its related variant gene editing system expression vector plasmid using the Golden gate test (FL101-01, TransGen Biotech).

[0092] The spcaer sequence of the ACTB human gene is specifically shown in SEQ ID NO.3, the spcaer sequence of the HSP90AA1 human gene is specifically shown in SEQ ID NO.4, the spcaer sequence of the DYNLT1 human gene is specifically shown in SEQ ID NO.5, the spcaer sequence of the EMX1 human gene is specifically shown in SEQ ID NO.6, the spcaer sequence of the VEGFA human gene is specifically shown in SEQ ID NO.7, and the spcaer sequence of the AAVS1 human gene is specifically shown in SEQ ID NO.8.

[0093] 2. Figure 1 The construction process of the MCP-RecE expression plasmid vector shown in B is as follows:

[0094] (1) The gene sequence of microbial-derived EcR RecE shown in SEQ ID NO. 17 was obtained by whole gene synthesis.

[0095] (2) The EF1A-MCP expression vector (shown in SEQ ID NO. 15) was digested with restriction endonucleases BamHI (FD0054, ThermoFisher) and EcoRI (FD0274, ThermoFisher) at 37°C for 40 min. The digested products were identified by 1.5% agarose gel electrophoresis and recovered.

[0096] (3) The EcR RecE gene sequence synthesized in (1) was Gibson ligated with the digestion product from step (2) at a molar ratio of 1:2. T5 exonuclease (2× MultiF Seamless Assembly Mix, Abclonal) and the product were mixed at a volume ratio of 1:1. The Gibson ligation reaction volume was 6 μl and the reaction was carried out at 50°C for 15 min.

[0097] The gene sequence of the MCP-RecE expression vector plasmid is shown in SEQ ID NO. 9. The amino acid sequence of MCP-RecE is shown in SEQ ID NO. 10.

[0098] Example 2 HDR repair template construction and amplification

[0099] Design of approximately 2kb HDR restoration template ( Figure 2 ), which contains an approximately 1 kb P2A-mKate fluorescent protein expression sequence (shown in SEQ ID NO. 11) and left and right 600 bp homology arm DNA sequences of the human genes ACTB, HSP90AA1, EMX1, VEGFA, and AAVS1. Only the left and right homology arm sequences of the DYNLT1 gene are 200 bp and 400 bp, respectively. The HDR repair template is amplified by PCR.

[0100] Among them, the HDR homologous repair template sequence of the ACTB human gene is shown in SEQ ID NO.12, the HDR homologous repair template sequence of the HSP90AA1 human gene is shown in SEQ ID NO.13, the HDR homologous repair template sequence of the DYNLT1 human gene is shown in SEQ ID NO.14, the HDR homologous repair template sequence of the EMX1 human gene is shown in SEQ ID NO.18, the HDR homologous repair template sequence of the VEGFA human gene is shown in SEQ ID NO.19, and the HDR homologous repair template sequence of the AAVS1 human gene is shown in SEQ ID NO.20.

[0101] Example 3 The spCas9-RecE gene editing system can effectively improve the efficiency of precise gene editing in HEK293T cells and improve the editing accuracy. The efficiency of precise editing of the spCas9-RecE gene editing system was evaluated in the HEK293T cell line.

[0102] The specific experimental process is as follows:

[0103] (1) Cell plating: HEK293T human cell line was cultured in DMEM (GS-3301, HomelandBio) complete medium supplemented with 10% FBS (GL-1001, HomelandBio) and 1% penicillin / streptomycin (BL505A, biosharp) at 37°C and 5% CO2. 14-16 h before transfection, when the confluence of HEK293T cells reached 80-90%, the supernatant was discarded, the cells were washed once with DPBS (C14190500BT, ThermoFisher), digested with 0.05%-Trypsin-EDTA (25200072, ThermoFisher), transferred to a 15 ml centrifuge tube, and centrifuged at 200 x g for 3 min. After centrifugation, the supernatant was discarded, the cells were resuspended in 1 ml of cell culture medium, and the cells were counted. HEK293T cells were cultured at a density of 7.0-8.0 × 10 4 Cells were plated in a 48-well plate (11410, Labselect) at a density of 100 μg / well and transfected when the cell confluence reached 60-70%.

[0104] (2) Cell transfection: Each 48-well cell was transfected with 10 ng of the HDR repair template DNA of the human gene HSP90AA1, DYNLT1, or AAVS1 prepared in Example 2 and 490 ng of plasmid DNA (including 350 ng of the MS2-sgRNA-spCas9 expression vector plasmid prepared in Step 1 of Example 1 and 140 ng of the MCP-RecE expression vector plasmid prepared in Step 2 of Example 1). TM (31985070, ThermoFisher) were diluted P3000 TM (L3000015, ThermoFisher) and Lipofectamine 3000 TM (L3000015, ThermoFisher). Each 48-well cell culture was treated with 12.5 μl Opti-MEM TM / P3000 TM Dilute 500 ng DNA in the premix and then add an equal volume of Opti-MEM TM / Lipofectamine 3000 TMLet the premix stand for 15 minutes. Add 25 μl of the mixed reagent to a single well of cells for transfection. Gently shake the 48-well plate to ensure even distribution of the reagents in the culture medium. 48 hours after transfection, replace the cell culture medium. 72 hours after transfection, harvest the cells for flow cytometry analysis.

[0105] (3) Flow cytometry analysis: After the HEK293T cells were treated in step (2), the HDR-mediated spCas9-RecE long-fragment gene precise editing efficiency was analyzed by flow cytometry, and the mKate positive rate represented the spCas9-RecE long-fragment gene precise editing efficiency. 72 hours after transfection, the supernatant was discarded, the cells were washed once with DPBS, and the cells were digested with 0.05%-Trypsin-EDTA. The cells in each well were transferred to a 1.5 ml EP tube and centrifuged at 200 x g for 3 minutes. After centrifugation, the supernatant was discarded and the cells were resuspended in DPBS containing 2% fetal bovine serum; BD FACSCelesta TM The results were analyzed using a cell analyzer (BD Biosciences).

[0106] (4) Fluorescence microscopy analysis: After HEK293T cells were treated in step (2) for 72 hours, an inverted fluorescence microscope (YHF40, Shanghai Yuehe) was used to photograph the mKate-positive cells in each transfection group. The mKate-positive rate represents the efficiency of precise editing of long-fragment genes by spCas9-RecE. In addition, control groups were set up as Blank: a blank control group without transfected DNA; NC: a negative control group without sgRNA targeting the gene site; and Cas9only: a negative control group transfected with MS2-sgRNA-spCas9 only and without transfection of MCP-RecE.

[0107] The results showed that EcR RecE could effectively mediate the expression of about 1 kb of exogenous P2A-mKate gene sequence in the HEK293T human cell genome HSP90AA1 ( Figure 3 A), DYNLT1( Figure 3 B) and AAVS1( Figure 3 C) Precise insertion of human gene loci, compared with Blank, NC and Cas9 only control groups. Fluorescence microscopy results showed that EcR RecE can effectively mediate the insertion of P2A-mKate gene sequence into HSP90AA1 gene locus ( Figure 3 D), compared with the Cas9 only control group.

[0108] Example 4: The spCas9-RecE gene editing system can effectively improve the efficiency of long-fragment gene editing in multiple eukaryotic cell lines

[0109] The spCas9-RecE gene editing system was used to insert P2A-mKate sequences into the HSP90AA1, AAVS1, and DYNLT1 human gene loci in Hela, A549, and HepG2 human cell lines. Flow cytometry was used to quantitatively assess the editing efficiency. The specific experimental procedures are as follows:

[0110] (1) Cell plating: Hela, A549, and HepG2 human cell lines were cultured in DMEM (GS-3301, Homeland Bio) supplemented with 10% FBS (GL-1001, Homeland Bio) and 1% penicillin / streptomycin (BL505A, Biosharp) at 37°C in a 5% CO2 atmosphere. 14–16 h before transfection, when the cells reached 80–90% confluence, the supernatant was discarded and the cells were washed once with DPBS (C14190500BT, Thermo Fisher). The cells were digested with 0.05%-Trypsin-EDTA (25200072, Thermo Fisher), transferred to a 15 ml centrifuge tube, and centrifuged at 200 × g for 3 min. After centrifugation, the supernatant was discarded and the cells were resuspended in 1 ml of DMEM complete medium for cell counting. Hela, A549 and HepG2 human cell lines were 3.0-5.0×10 4 Cells were plated in a 48-well plate (11410, Labselect) at a density of 100 μg / well and transfected when the cell confluence reached 60-70%.

[0111] (2) Cell transfection: Each 48-well cell was transfected with 10 ng of the HDR repair template DNA of the human gene HSP90AA1, DYNLT1, or AAVS1 prepared in Example 2 and 490 ng of plasmid DNA (including 350 ng of the MS2-sgRNA-spCas9 expression vector plasmid prepared in Step 1 of Example 1 and 140 ng of the MCP-RecE expression vector plasmid prepared in Step 2 of Example 1). TM (31985070, ThermoFisher) were diluted P3000 TM (L3000015, ThermoFisher) and Lipofectamine 3000 TM (L3000015, ThermoFisher). Each 48-well cell culture was treated with 12.5 μl Opti-MEM TM / P3000 TM Dilute 500 ng DNA in the premix and then add an equal volume of Opti-MEM TM / Lipofectamine 3000 TMLet the premix stand for 15 minutes. Add 25 μl of the reagent mixture to a single well of cells for transfection. Gently shake the 48-well plate to ensure even distribution of the mixture throughout the culture medium. 48 hours after transfection, replace the cell culture medium. 72 hours after transfection, harvest the cells for analysis.

[0112] (3) Flow cytometry analysis: After Hela, A549, and HepG2 human cells were treated in step (2), the HDR-mediated spCas9-RecE long-fragment gene precise editing efficiency was analyzed by flow cytometry, and the mKate positive rate represented the spCas9-RecE long-fragment gene precise editing efficiency. 72 hours after transfection, the supernatant was discarded, the cells were washed once with DPBS, and the cells were digested with 0.05%-Trypsin-EDTA. The cells in each well were transferred to the corresponding cell culture wells of a U-shaped 96-well plate and centrifuged at 200xg for 3 minutes. After centrifugation, the supernatant was discarded and the cells were resuspended in DPBS containing 2% fetal bovine serum; BD FACSCelesta TM The results were analyzed using a cell analyzer (BD Biosciences).

[0113] The control groups were NC: negative control group without gene site targeting sgRNA; Cas9 only: negative control group with only MS2-sgRNA-spCas9 transfection and no MCP-RecE transfection.

[0114] Flow cytometry results showed that the spCas9-RecE gene editing system could be used in the AAVS1 human gene locus of A549 human cell line ( Figure 4 A left), DYNLT1 human gene locus ( Figure 4 A right), DYNLT1 human gene locus of HepG2 human cell line ( Figure 4 B left), HSP90AA1 human gene site ( Figure 4 B right) and DYNLT1 human gene locus of Hela human cell line ( Figure 4 C left), HSP90AA1 human gene locus ( Figure 4 C (right) mediated precise insertion of an approximately 1 kb P2A-mKate exogenous fragment, with an insertion efficiency significantly higher than that of the Cas9 only control group.

[0115] Example 5: The spCas9-RecE gene editing system can effectively reduce safety risks such as off-target effects

[0116] 1. Design iGuide and NGS sequencing to evaluate the random integration efficiency of the RecE genome editing system constructed in Example 1 to ensure that the system does not increase the occurrence of random integration events.

[0117] The specific operations are:

[0118] (1) Cell plating: HEK293T cells were cultured in DMEM (GS-3301, HomelandBio) complete medium supplemented with 10% FBS (GL-1001, HomelandBio) and 1% penicillin / streptomycin (BL505A, biosharp) at 37°C in 5% CO2. 14-16 hours before transfection, when the cell confluence reached 80-90%, the supernatant was discarded, the cells were washed once with DPBS (C14190500BT, ThermoFisher), digested with 0.05%-Trypsin-EDTA (25200072, ThermoFisher), transferred to a 15 ml centrifuge tube, and centrifuged at 200 x g for 3 min. After centrifugation, the supernatant was discarded, the cells were resuspended in 1 ml DMEM complete medium, and the cells were counted. 3.0-5.0 × 10 4 Cells were plated in a 48-well plate (11410, Labselect) at a density of 100 μg / well and transfected when the cell confluence reached 60-70%.

[0119] (2) Cell transfection: Each 48-well cell was transfected with 490 ng of plasmid DNA (including 350 ng of the MS2-sgRNA-spCas9 expression vector plasmid prepared in step 1 of Example 1 and 140 ng of the MCP-RecE expression vector plasmid prepared in step 2 of Example 1). TM (31985070, ThermoFisher) were diluted P3000 TM (L3000015, ThermoFisher) and Lipofectamine3000 TM (L3000015, ThermoFisher). Each 48-well cell culture was treated with 12.5 μl Opti-MEM TM / P3000 TM Dilute 490ng DNA in the premix and then add an equal volume of Opti-MEM TM / Lipofectamine3000 TM Let the premix stand for 15 minutes. Add 25 μl of the mixed reagent to a single well of cells for transfection, gently shaking the 48-well plate to ensure even distribution of the reagents in the culture medium. 48 hours after transfection, replace the cell culture medium. 72 hours after transfection, harvest the cells and extract genomic DNA using a genomic DNA extraction kit (Qiagen, Catalog #51306).

[0120] (3) PCR amplification: The genomic DNA extracted in step (2) was amplified using Taq enzyme (Takara, catalog number #R004A). The reaction system was as follows: 2X Taq premix: 10 μl; universal PCR primer (polyT sequence): 2 μl; genomic DNA: 1 μl; water: 7 μl. The reaction procedure was as follows: 98°C, 5 seconds; 98°C, 10 seconds; 72°C, 30 seconds (repeat 2 and 3 for 25 cycles); 72°C, 10 minutes; and storage at 4°C.

[0121] After the PCR reaction was completed, the PCR product was purified using a PCR product purification kit (Qiagen, catalog number #28104) and subjected to next-generation sequencing.

[0122] (4) Next-generation sequencing and data analysis: The PCR products were prepared for sequencing, the concentration was determined, and 10 ng was added to the Illumina MiSeq Reagent Kit v3 and sequenced on an Illumina Miseq sequencer. After sequencing, the results were uploaded to the open-source data analysis tool CRISPResso2 (http: / / crispresso2.pinellolab.org / submission) for data analysis.

[0123] 2. The off-target efficiency of spCas9-RecE mediated by Example 1 was evaluated. The off-target sites were predicted in the genome according to the sgRNA targeting sequence, and PCR primers were designed to amplify and sequence the sites by NGS to ensure that the system did not increase the off-target risk of spCas9.

[0124] The specific operations are:

[0125] (1) Cell plating: HEK293T cells were cultured in DMEM (GS-3301, HomelandBio) complete medium supplemented with 10% FBS (GL-1001, HomelandBio) and 1% penicillin / streptomycin (BL505A, biosharp) at 37°C in 5% CO2. 14-16 hours before transfection, when the cell confluence reached 80-90%, the supernatant was discarded, the cells were washed once with DPBS (C14190500BT, ThermoFisher), digested with 0.05%-Trypsin-EDTA (25200072, ThermoFisher), transferred to a 15 ml centrifuge tube, and centrifuged at 200 x g for 3 min. After centrifugation, the supernatant was discarded, the cells were resuspended in 1 ml DMEM complete medium, and the cells were counted. 3.0-5.0 × 10 4Cells were plated in a 48-well plate (11410, Labselect) at a density of 100 μg / well and transfected when the cell confluence reached 60-70%.

[0126] (2) Cell transfection: Each 48-well cell was transfected with 490 ng of plasmid DNA (including 350 ng of the MS2-sgRNA-spCas9 expression vector plasmid prepared in step 1 of Example 1 and 140 ng of the MCP-RecE expression vector plasmid prepared in step 2 of Example 1). TM (31985070, ThermoFisher) were diluted P3000 TM (L3000015, ThermoFisher) and Lipofectamine3000 TM (L3000015, ThermoFisher). Each 48-well cell culture was treated with 12.5 μl Opti-MEM TM / P3000 TM Dilute 490ng DNA in the premix and then add an equal volume of Opti-MEM TM / Lipofectamine3000 TM Let the premix stand for 15 minutes. Add 25 μl of the mixed reagent to a single well of cells for transfection, gently shaking the 48-well plate to ensure even distribution of the reagents in the culture medium. 48 hours after transfection, replace the cell culture medium. 72 hours after transfection, harvest the cells and extract genomic DNA using a genomic DNA extraction kit (Qiagen, Catalog #51306).

[0127] (3) PCR amplification: The genomic DNA extracted in step (2) was amplified using Taq enzyme (Takara, catalog number #R004A). The reaction system was as follows: 2X Taq premix: 10 μl; specific PCR primers: 2 μl; genomic DNA: 1 μl; water: 7 μl. The reaction procedure was as follows: 98°C, 5 seconds; 98°C, 10 seconds; 72°C, 30 seconds (repeat 2 and 3 for 25 cycles); 72°C, 10 minutes; and storage at 4°C.

[0128] The EMX1 PCR primer information is as follows:

[0129] Forward: 5'-CACCGTCACCTCCAATGACTAGGG-3'

[0130] Reverse: 5'-AAACCCCTAGTCATTGGAGGTGAC-3'

[0131] The VEGFA PCR primer information is as follows:

[0132] Forward: 5'-CACCCCATTCACCCAGCTTCCCTG-3'

[0133] Reverse: 5'-AAACCAGGGAAGCTGGGTGAATGG-3'

[0134] After the PCR reaction was completed, the PCR product was purified using a PCR product purification kit (Qiagen, catalog number #28104) and subjected to next-generation sequencing.

[0135] (4) Next-generation sequencing and data analysis: The PCR products were prepared for sequencing, the concentration was determined, and 10 ng was added to the Illumina MiSeq Reagent Kit v3 and sequenced on an Illumina Miseq sequencer. After sequencing, the results were uploaded to the open-source data analysis tool CRISPResso2 (http: / / crispresso2.pinellolab.org / submission) for data analysis.

[0136] A Cas9 only control group was set up: only MS2-sgRNA-spCas9 was transfected, and no MCP-RecE was transfected into the negative control group.

[0137] The experimental results showed that EcR RecE overexpression did not increase the expression of spCas9 in the EMX1 human gene locus ( Figure 5 A) and VEGFA human gene locus ( Figure 5 B) Off-target efficiency. The Guide-seq structure of the EMX1 human gene locus and the VEGFA human gene locus showed that the off-target efficiency of the EcR RecE group was similar to or lower than that of the control group in the whole genome ( Figure 5 C). The statistical results of chromosome off-target editing show that EcR RecE overexpression does not increase the off-target efficiency of spCas9 ( Figure 5 D).

[0138] Example 6: The spCas9(D10A) / spCas9(H840A) / dCas9-RecE gene editing system can effectively improve the efficiency of long-fragment gene editing at multiple sites in HEK293T cells

[0139] EcR RecE was applied to two different nickase systems, spCas9(D10A) and spCas9(H840A), and the dCas9 system. The precise editing efficiency of spCas9(D10A) / spCas9(H840A) / dCas9-RecE was evaluated at the HSP90AA1 and DYNLT1 human gene loci in HEK293T cell lines. Flow cytometry was used to quantitatively analyze the insertion efficiency of the approximately 1kb P2A-mKate exogenous long fragment. The specific experimental process is as follows:

[0140] (1) Cell plating: HEK293T human cell line was cultured in DMEM (GS-3301, HomelandBio) complete medium supplemented with 10% FBS (GL-1001, HomelandBio) and 1% penicillin / streptomycin (BL505A, biosharp) at 37°C and 5% CO2. 14-16 h before transfection, when the confluence of HEK293T cells reached 80-90%, the supernatant was discarded, the cells were washed once with DPBS (C14190500BT, ThermoFisher), digested with 0.05%-Trypsin-EDTA (25200072, ThermoFisher), transferred to a 15 ml centrifuge tube, and centrifuged at 200 x g for 3 min. After centrifugation, the supernatant was discarded, the cells were resuspended in 1 ml DMEM complete medium, and the cell count was performed. HEK293T cells were expressed at 7.0-8.0 × 10 4 Cells were plated in a 48-well plate (11410, Labselect) at a density of 100 μg / well and transfected when the cell confluence reached 60-70%.

[0141] (2) Cell transfection: Each 48-well cell was transfected with 10 ng of the HDR repair template DNA of the human gene HSP90AA1, DYNLT1, or AAVS1 prepared in Example 3 and 490 ng of plasmid DNA (including 350 ng of the MS2-sgRNA-spCas9(D10A) / spCas9(H840A) / dCas9 expression vector plasmid prepared in Step 1 of Example 1, and 140 ng of the MCP-RecE expression vector plasmid or 140 ng of the MCP-RecT expression vector plasmid prepared in Step 2 of Example 1). TM (31985070, ThermoFisher) were diluted P3000 TM (L3000015, ThermoFisher) and Lipofectamine 3000 TM (L3000015, ThermoFisher). Each 48-well cell culture was treated with 12.5 μl Opti-MEMTM / P3000 TM Dilute 500 ng DNA in the premix and then add an equal volume of Opti-MEM TM / Lipofectamine 3000 TM Let the premix stand for 15 minutes. Add 25 μl of the reagent mixture to a single well of cells for transfection. Gently shake the 48-well plate to ensure even distribution of the mixture throughout the culture medium. 48 hours after transfection, replace the cell culture medium. 72 hours after transfection, harvest the cells for analysis.

[0142] (3) Flow cytometry analysis: After HEK293T cells were treated with steps (1-2), the HDR-mediated spCas9 (D10A) / spCas9 (H840A) / dCas9-RecE long-fragment gene precise editing efficiency was analyzed by flow cytometry. The mKate positive rate represents the spCas9 (D10A) / spCas9 (H840A) / dCas9-RecE long-fragment gene precise editing efficiency. 72h after transfection, the supernatant was discarded, the cells were washed once with DPBS, and 0.05%-Trypsin-EDTA was used to digest the cells. The cells in each well were transferred to the corresponding cell culture wells of a U-shaped 96-well plate and centrifuged at 200xg for 3min. After centrifugation, the supernatant was discarded and the cells were resuspended in DPBS containing 2% FBS; BD FACSCelesta TM The results were analyzed using a cell analyzer (BD Biosciences).

[0143] The control groups were NC: negative control group with no gene site targeting sgRNA; nCas9 only: transfection of MS2-sgRNA-spCas9(D10A) / spCas9(H840A) / dCas9: negative control group with no transfection of MCP-RecE; SSAP / RecT group: positive control group with spCas9(D10A) / spCas9(H840A) / dCas9-RecT (the plasmid vector construction method of this group can be found in DOI:10.1093 / nar / gkaa1264 and DOI:10.1038 / s41556-021-00836-1).

[0144] The efficiency of editing exogenous long fragments was quantitatively analyzed by flow cytometry. The results showed that RecE was applied to spCas9(D10A)( Figure 6 A), spCas9(H840A) Figure 6 A) and dCas9( Figure 6B) The system can effectively improve the efficiency of long-fragment gene editing in HEK293T cells. The editing efficiency of the EcR RecE group was significantly higher than that of the nCas9 only control group.

[0145] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of microbial protein RecE in constructing a gene editing system, characterized in that: The microbial protein RecE can improve the efficiency of long-fragment gene editing mediated by the CRISPR / Cas9 gene editing system in eukaryotic cells. Preferably, the nucleotide sequence of microbial-derived RecE is shown in SEQ ID NO.

17.

2. A CRISPR / Cas9 gene editing system based on the microbial protein RecE, characterized in that: The gene editing system consists of the following three elements: MS2-sgRNA-Cas9 gene editing system expression vector, MCP-RecE expression vector and HDR homology repair template.

3. The gene editing system according to claim 2, characterized in that The MCP-RecE expression vector includes an MCP-RecE sequence, which is a fusion expression of the microbial protein RecE and the MCP aptamer protein; preferably, the base vector of the MCP-RecE expression vector is the EF1A-MCP expression vector, and the RecE sequence is inserted between the BamHI and EcoRI restriction sites of the EF1A-MCP expression vector to construct it; further preferably, the sequence of the EF1A-MCP expression vector is shown in SEQ ID NO.

15.

4. The gene editing system according to claim 3, characterized in that The gene sequence of the MCP-RecE expression vector is shown in SEQ ID NO.9, and the amino acid sequence of MCP-RecE is shown in SEQ ID NO.

10.

5. The gene editing system according to claim 2, characterized in that The MS2-sgRNA-Cas9 gene editing system expression vector includes an MS2-sgRNA sequence and a Cas9 nuclease, the MS2-sgRNA sequence includes an MS2-sgRNAscaffold sequence, and the Cas9 nuclease is selected from spCas9 or a related variant sequence thereof; preferably, the spCas9-related variant in the MS2-sgRNA-Cas9 gene editing system expression vector is selected from spCas9 (D10A), spCas9 (H840A) or dCas9; preferably, the expression vector further comprises a T2A-EBFP sequence.

6. The gene editing system according to claim 5, characterized in that The basic vector of the MS2-sgRNA-Cas9 gene editing system expression vector is the pU6-CBh-T2A-EBFP expression vector, and the nucleotide sequence of the pU6-CBh-T2A-EBFP expression vector is shown in SEQ ID NO.16; the MS2-sgRNA-Cas9 gene editing system expression vector is constructed by connecting the MS2-sgRNA scaffold sequence between the NdeI restriction site of the basic vector and the Cas9 nuclease sequence between the AgeI and HindIII restriction sites.

7. The gene editing system according to claim 5, characterized in that When the Cas9 nuclease in the MS2-sgRNA-Cas9 gene editing system expression vector is spCas9, the gene sequence of the MS2-sgRNA-spCas9 gene editing system expression vector is shown in SEQ ID NO.1; when the Cas9 nuclease in the MS2-sgRNA-Cas9 gene editing system expression vector is dCas9, the gene sequence of the MS2-sgRNA-dCas9 gene editing system expression vector is shown in SEQ ID NO.

2.

8. The gene editing system according to claim 5, characterized in that The MS2-sgRNA-dCas9 gene editing system expression vector further includes a spacer sequence targeting the gene site to be edited; the spacer sequence targeting the gene site to be edited is connected between the BbsI restriction sites of the MS2-sgRNA scaffold sequence in the MS2-sgRNA-Cas9 gene editing system expression vector shown in SEQ ID NO.1 or SEQ ID NO.2; Preferably, the gene to be edited is selected from the group consisting of ACTB human gene, HSP90AA1 human gene, DYNLT1 human gene, EMX1 human gene, VEGFA human gene, and AAVS1 human gene; Further preferably, the spcaer sequence of the ACTB human gene is specifically shown as SEQ ID NO.3, the spcaer sequence of the HSP90AA1 human gene is specifically shown as SEQ ID NO.4, the spcaer sequence of the DYNLT1 human gene is specifically shown as SEQ ID NO.5, the spcaer sequence of the EMX1 human gene is specifically shown as SEQ ID NO.6, the spcaer sequence of the VEGFA human gene is specifically shown as SEQ ID NO.7, and the spcaer sequence of the AAVS1 human gene is specifically shown as SEQ ID NO.

8.

9. The gene editing system according to claim 2, characterized in that The HDR homology repair template includes a P2A-mKate fluorescent protein expression sequence and a homology arm DNA sequence of the gene to be edited located on the left and right sides of the P2A-mKate fluorescent protein expression sequence, wherein the P2A-mKate fluorescent protein expression sequence is shown in SEQ ID NO.11; Preferably, the gene to be edited is selected from the group consisting of ACTB human gene, HSP90AA1 human gene, DYNLT1 human gene, EMX1 human gene, VEGFA human gene, and AAVS1 human gene; Further preferably, except for the DTNLT1 human gene, the DNA sequences of the left homologous arms and right homologous arms of the other genes to be edited are both 600 bp or more, and the DNA sequences of the left and right homologous arms of the DYNLT1 human gene are 200 bp and 400 bp or more, respectively; Further preferably, the homologous repair template sequence of the ACTB human gene is shown as SEQ ID NO.12, the homologous repair template sequence of the HSP90AA1 human gene is shown as SEQ ID NO.13, the homologous repair template sequence of the DYNLT1 human gene is shown as SEQ ID NO.14, the HDR homologous repair template sequence of the EMX1 human gene is shown as SEQ ID NO.18, the HDR homologous repair template sequence of the VEGFA human gene is shown as SEQ ID NO.19, and the HDR homologous repair template sequence of the AAVS1 human gene is shown as SEQ ID NO.

20.

10. Use of the gene editing system according to claim 2 in the preparation of drugs or reagents for cell therapy and / or gene therapy-related diseases; Preferably, the cell therapy is based on HEK293T cells, A549 cells, HepG2 cells or Hela cell lines, and the application can improve the efficiency of long-fragment gene editing at multiple sites in cells; Further preferably, the disease is autosomal dominant polycystic kidney disease, autosomal recessive hepatolenticular degeneration, sickle cell disease or β-thalassemia, Duchenne muscular dystrophy, congenital amaurosis, or cervical cancer.

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