Exonuclease-mediated long-fragment precise gene editing system

Abstract

The invention belongs to the technical field of biological medicine, a gene editing system MMEJ-Exo is constructed through humanized application of exonuclease or key functional domains derived from microorganisms and eukaryotes, spCas9-mediated eukaryote cells are remarkably improved, especially long-fragment editing of primary neuronal cells is remarkably improved, and the gene editing efficiency is improved. And a new technology and a new method are provided for gene therapy and cell therapy transformation application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. It constructs a gene editing system, MMEJ-Exo, by using humanized exonucleases or key functional domains derived from microorganisms and eukaryotes. This system significantly improves long-fragment editing of eukaryotic cells, especially primary neurons, mediated by spCas9, providing new technologies and methods for gene therapy and cell therapy translational applications. Background Technology

[0002] For major diseases such as those related to gene mutations, gene therapy and cell therapy strategies based on CRISPR / Cas9 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 Casgevy, the world's first CRISPR / Cas9 cell therapy product, for the treatment of sickle cell disease, a hereditary blood disorder, marking the beginning of gene therapy's 2.0 era.

[0003] There are over 7,000 rare diseases worldwide, affecting more than 300 million people. 80% of these are caused by polymorphic mutations in single genes, including single-base and long-segment alterations in genetic information. However, current gene therapy strategies primarily involve knocking out the pathogenic mutated gene, rather than precisely repairing the pathogenic mutation (especially long-segment DNA deletions or duplications), which limits its clinical translational application to some extent. Therefore, the development of safe, precise, and efficient long-segment (kilobase level) gene editing tools has become a key factor in promoting the further development of gene therapy translational applications.

[0004] MMEJ (Microhomology-Mediated End Joining) is a novel gene editing strategy that uses microhomology arms (5-25 bp) to mediate alternative end joining for repairing broken DNA. Compared to NHEJ and HDR, MMEJ offers the following advantages as a novel gene editing strategy: 1) HDR uses homologous sequences to introduce DSB damage sites into the target gene for precise repair. However, because the action of homologous fragments on the target gene site depends on the binding of DNA fragments hundreds of bases upstream and downstream of the target gene, its gene editing efficiency is the lowest. Furthermore, constructing HDR homologous sequences is time-consuming, labor-intensive, and costly. In contrast, the short homologous arms required for MMEJ can be added using PCR, which is simple and efficient. 2) MMEJ's editing efficiency is between NHEJ and HDR, and its micro-homogeneous arms can ensure the accurate insertion of foreign fragments; 3) In addition, compared to HDR, which only works on cells in the dividing phase because it acts on the G2 / S phase, MMEJ is applicable to the vast majority of cells, regardless of whether they are in an active dividing phase. 4) HDR-mediated exogenous fragment insertion accuracy is limited. As the length of the inserted fragment increases, the editing efficiency decreases significantly, while the editing efficiency of MMEJ is less affected by the length. Summary of the Invention

[0005] This patent successfully created a novel gene editing system, MMEJ-Exo, by applying humanized exonucleases or key functional domains derived from microorganisms and eukaryotes. This significantly improves the efficiency of long-fragment editing of eukaryotic genomes mediated by spCas9, especially in non-dividing primary neurons.

[0006] In this invention, MMEJ refers to pico-homological end repair; Exo refers to exonuclease; and long fragment refers to a level of nearly 1,000 bases.

[0007] The RecE protein described in this invention is officially named PadRecE. The key functional domain of the eukaryotic CtIP is the N-terminal CtIP nuclease functional domain (hereinafter abbreviated as CtIP_N).

[0008] The purpose of this invention is to provide a series of novel long-fragment gene editing systems based on the MMEJ repair principle, which can realize precise, safe, and efficient long-fragment gene editing operations on eukaryotic genomes, especially to confirm the gene editing efficacy in primary neurons, and provide effective application tools for in vivo and in vitro gene therapy and cell therapy for gene mutation-related diseases, thereby promoting the development of clinical translational applications.

[0009] The technical solution of this invention is as follows: The first objective of this invention is to provide the application of the combination of the microbial protein RecE and the key functional domain CtIP_N of the eukaryotic protein CtIP in the construction of a gene editing system, wherein the gene editing system is mediated by the CRISPR / Cas9 gene editing system in eukaryotic cells, and the efficiency of long-fragment gene editing mediated by the gene editing system is improved by the combination of the microbial protein RecE and the key functional domain CtIP_N of the eukaryotic protein CtIP. Preferably, the nucleotide sequence of the microbial RecE is shown in SEQ ID NO.1, and the nucleotide sequence of the key functional domain CtIP_N of the eukaryotic protein CtIP is shown in SEQ ID NO.2.

[0010] A second objective of this invention is to provide an MMEJ-Exo gene editing system based on exonuclease, wherein the gene editing system comprises the following three components: (1) MCP-RecE expression vector and MCP-CtIP_N expression vector; (2) MS2-sgRNA-spCas9 gene editing system expression vector; (3) MMEJ homology repair template.

[0011] Furthermore, (1) the MCP-RecE expression vector includes the MCP-RecE sequence, wherein the MCP-RecE sequence is a fusion expression of microbial protein RecE and MCP aptamer protein; the MCP-CtIP_N expression vector includes the MCP-CtIP_N sequence, wherein the MCP-CtIP_N sequence is a fusion expression of eukaryotic protein CtIP key functional domain CtIP_N and MCP aptamer protein; Preferably, the amino acid sequence of the MCP-RecE is shown in SEQ ID NO.4; and the amino acid sequence of the MCP-CtIP_N is shown in SEQ ID NO.5.

[0012] Preferably, (1) the base vector of the MCP-RecE expression vector and the MCP-CtIP_N expression vector is a plasmid containing the MCP aptamer protein; When the key functional domain CtIP_N of the microbial protein RecE and the eukaryotic protein CtIP is incorporated into the basic vector, it is expressed by fusion with the MCP aptamer protein inherent in the basic vector.

[0013] More preferably, the plasmid containing the MCP aptamer protein is an EF1A-MCP expression vector, and the MCP-RecE expression vector and the MCP-CtIP_N expression vector are constructed by inserting the RecE sequence of the microbial protein or the CtIP_N sequence of the key functional domain of the CtIP of the eukaryotic protein between the BamHI and EcoRI restriction sites of the EF1A-MCP expression vector. More preferably, the sequence of the EF1A-MCP expression vector is shown in SEQ ID NO.3.

[0014] Furthermore, the amino acid sequence of the MCP-RecE is shown in SEQ ID NO.4; the amino acid sequence of the MCP-CtIP_N is shown in SEQ ID NO.5.

[0015] Furthermore, (2) the expression vector of the MS2-sgRNA-spCas9 gene editing system includes the MS2-sgRNAscaffold sequence and the spCas9 nuclease; Preferably, the expression vector further comprises a T2A-EBFP sequence for evaluating cell transfection efficiency.

[0016] Furthermore, the base vector of the MS2-sgRNA-spCas9 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.6; the MS2-sgRNA-spCas9 gene editing system expression vector is constructed by ligating the MS2-sgRNA scaffold sequence between the NdeI restriction sites of the base vector and ligating the spCas9 nuclease sequence between the AgeI and HindIII restriction sites, and the MS2-sgRNA scaffold sequence is shown in SEQ ID NO.19.

[0017] Furthermore, when the Cas9 nuclease in the MS2-sgRNA-spCas9 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.7.

[0018] Furthermore, the MS2-sgRNA-spCas9 gene editing system expression vector also includes a spacer sequence that targets the gene site to be edited; Preferably, the spacer sequence targeting the gene site to be edited is inserted between the BbsI restriction sites of the MS2-sgRNAscaffold sequence shown in SEQ ID NO.19; Preferably, the gene to be edited is selected from the ACTB human gene, HSP90AA1 human gene, CLTA human gene, SEC61B human gene, and ACTB mouse gene. Further preferred embodiments include the spacer sequence targeting the human ACTB gene as shown in SEQ ID NO. 8, the spacer sequence targeting the human HSP90AA1 gene as shown in SEQ ID NO. 9, the spacer sequence targeting the human CLTA gene as shown in SEQ ID NO. 10, the spacer sequence targeting the human SEC61B gene as shown in SEQ ID NO. 11, and the spacer sequence targeting the mouse ACTB gene as shown in SEQ ID NO. 12.

[0019] Based on the working principle of the SAM recruitment system, the MCP aptamers of the MCP-RecE and MCP-CtIP_N expression vectors specifically bind to the MS2 stem-loop structure of the MS2-sgRNA-spCas9 gene editing system expression vector, enabling RecE or CtIP_N to synergistically perform gene editing functions with sgRNA-spCas9 in eukaryotic cells.

[0020] Furthermore, (3) the MMEJ homology repair template includes a P2A-mKate fluorescent protein expression sequence and a homologous arm DNA sequence of the gene to be edited located to the left and right of the P2A-mKate fluorescent protein expression sequence, as shown in SEQ ID NO.13; Preferably, the DNA sequences of the left and right homologous arms of the gene to be edited are optionally 5-25 bp; Preferably, the gene to be edited is selected from the ACTB human gene, HSP90AA1 human gene, CLTA human gene, SEC61B human gene, and ACTB mouse gene. Further preferred embodiments include the MMEJ homology repair template sequence of the ACTB human gene as shown in SEQ ID NO.14, the MMEJ homology repair template sequence of the HSP90AA1 human gene as shown in SEQ ID NO.15, the MMEJ homology repair template sequence of the CLTA human gene as shown in SEQ ID NO.16, the MMEJ homology repair template sequence of the SEC61B human gene as shown in SEQ ID NO.17, and the MMEJ homology repair template sequence of the ACTB mouse gene as shown in SEQ ID NO.18.

[0021] A third objective of this invention is to provide the application of the aforementioned gene editing system in the preparation of drugs or reagents for diseases related to cell therapy and / or gene therapy; Preferably, the cell therapy is based on one or more of the HEK293T cell line, HeLa cell line, MCF7 cell line, and primary neurons; The gene therapy is based on one or more of the ACTB gene, HSP90AA1 gene, CLTA gene, and SEC61B gene; The application can improve the efficiency of long-fragment gene editing at one or more sites in cells; More preferably, the disease is autosomal dominant polycystic kidney disease (ADPKD), cervical cancer, or breast cancer.

[0022] The precise, safe, and efficient gene editing mediated by the MMEI-Exo gene editing system of this invention is mainly achieved through the following steps: 1. Cell culture: Culture conditions for HEK293T and other human cell lines: High glucose medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U / ml penicillin and 100 μg / ml streptomycin, was used for culture in a 5% CO2, 37°C constant temperature cell culture incubator.

[0023] 2. Cell resuscitation: Remove the cells to be revived from liquid nitrogen and quickly place them in 37°C warm water to thaw. After complete thawing, transfer the cryopreservation solution to 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, 100 U / ml penicillin, and 100 μg / ml streptomycin), and incubate at 37°C under saturated humidity, 5% CO2. Change the medium after the cells have adhered to the dish.

[0024] 3. Cell passage: Once the cell confluence reaches 80-90%, discard the culture supernatant, wash once with DPBS, add 0.05% Trypsin-EDTA, and incubate at 37°C for 1 min. When the cells become spherical and slightly detach, add DMEM culture medium containing 10% fetal bovine serum to stop the digestion. Then, transfer the cell suspension to a 15 ml centrifuge tube and centrifuge at 200 x g for 3 min. After centrifugation, discard the supernatant, resuspend the cells in 1 ml of complete DMEM culture medium, and passage according to the required ratio.

[0025] 4. Cell cryopreservation: Once the cell confluence reached 95%, discard the culture medium 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 min. After centrifugation, discard the supernatant and resuspend the cells in the prepared cell cryopreservation solution (DMEM: FBS: DMSO = 7:2:1). Transfer the cell cryopreservation solution containing cells to a 2ml cryovial and incubate overnight at -80℃, then transfer to liquid nitrogen for storage.

[0026] 5. Cell transfection: 14-16 hours before transfection, HEK293T cells were injected at a rate of 7.0-8.0 × 10⁶ cells / year. 4 Cells were seeded at a density of 60-70% in 48-well plates and transfected after the cells reached 60-70% confluence. Pre-treatment with Opti-MEM was performed. TM P3000 diluted separately TM and Lipofectamine 3000 TM Each well contains 48 cells, and 12.5 μl of Opti-MEM was used. TM / P3000 TM Dilute 500 ng of plasmid DNA with the premixed buffer, then add an equal volume of Opti-MEM. TM Lipofectamine 3000 TMPremixed solution, let stand for 15 min. Add 25 μl of the mixed transfection reagent to each well for transfection, gently agitate the 48-well plate to ensure even distribution in the culture medium. Replace the cell culture medium 48 h after transfection. Collect cells 72 h after transfection for subsequent experimental analysis. HeLa and MCF7 cells were cultured at 5–6.0 × 10⁻⁶ cells per well. 4 and 3~4×10 4 Cells were seeded at a density of 500 ng DNA plasmid per well in 48-well plates. The cell culture medium was changed at 6 h and 48 h post-transfection. Cells were collected 72 h post-transfection for subsequent experimental analysis.

[0027] 6. Flow cytometry analysis: The insertion efficiency of mKate or EGFP fluorescent proteins was quantitatively analyzed by flow cytometry. 72 h post-transfection, the supernatant was discarded, and the cells were washed once with DPBS and digested with 0.05% Trypsin-EDTA. Cells were transferred to 1.5 ml EP tubes from each well and centrifuged at 200 x g for 3 min. After centrifugation, the supernatant was discarded, and the cells were resuspended in DPBS containing 2% fetal bovine serum. Gene editing efficiency was analyzed by flow cytometry within 2 hours.

[0028] 7. Extraction of primary neurons from mice: Cortical and hippocampal neurons were obtained from C57BL / 6J mice at embryonic day 16 (E16). Briefly, pregnant mice were anesthetized with isoflurane, embryos (both male and female) were rapidly removed, and the mothers were euthanized by decapitation. Cortical and hippocampal tissues were then isolated and kept viable in ice-cold dissection buffer, followed by digestion with 0.25% trypsin-EDTA in an incubator for 15 minutes. After terminating the enzymatic reaction with fetal bovine serum, the tissues were gently dissociated into a single-cell suspension using a flame-polished pipette tip. Neurons were centrifuged at low speed (100 g, 5 min) and resuspended in culture medium. Cell density was determined using a cell counter, and the required number of cells (4 × 10⁶ cells per sample) were collected according to experimental needs. 6 (1 cell), then centrifuge again at low speed.

[0029] 8. Electrotransmission of primary mouse neurons: Neurons were resuspended in nuclear transfection medium (Lonza) and 5 μg of plasmid DNA was added. The cell / DNA suspension was transferred to a dedicated electroporation cuvette and electroporated according to the O-005 program. Electroporated cells were seeded into six-well plates pre-coated with poly-D-lysine (0.05 mg / mL; Sigma-Aldrich) and replaced with fresh Neurobasal Plus medium to remove cell debris. Primary neurons were cultured for 10 days, with half the medium replaced every 4 days. On day 10, mKate-positive neurons were imaged using a fluorescence microscope. The entire culture was performed in a humidified incubator at 37 °C and 5% CO2.

[0030] The key components of the MMEJ-Exo gene editing system, MCP-RecE, MCP-CtIP_N, and MS2-sgRNA-spCas9 expression vector, are mainly achieved through the following steps: 9. Plasmid construction: The RecE, CtIP_N, and MS2-sgRNA-spCas9 gene sequences were obtained through whole-gene synthesis (in some specific embodiments, sequence information is shown in SEQ ID NO. 1, 2, and 7). RecE and CtIP_N were ligated into the EFlA-MCP expression vector plasmid using a Gibson assay, or sRNA-MS2-spCas9 was ligated into the hU6-CBh-T2A-EBFP expression vector plasmid. After the Gibson assay, the products were placed on ice for E. coli competent cell transformation experiments.

[0031] 10. Plasmid transformation: Thaw competent E. coli cells on ice. Once completely thawed, add the plasmid to be transformed into competent E. coli cells at a 1:10 volume ratio, gently mix with a pipette tip, and incubate on ice for 10 minutes. Streak the competent cell mixture onto LB agar plates containing ampicillin and incubate at 37°C for 12–18 hours.

[0032] 11. Plasmid extraction: After single colonies have 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 culture and extract plasmids. The specific experimental steps are as follows: (1) Take 4 ml of bacterial culture. Centrifuge at 12,000 rpm for 30 s, discard the supernatant, and collect the bacterial precipitate.

[0033] (2) Add 250 μl of solution P1 to resuspend the bacterial precipitate and vortex until completely suspended.

[0034] (3) Add 250 μl of solution P2, gently invert 6 times to fully lyse the cells, and let stand at room temperature for 4 min.

[0035] (4) Add 350 μl of solution P3 and immediately gently invert it 6 times; after a white flocculent precipitate appears, centrifuge at 12,000 rpm for 10 min.

[0036] (5) After centrifugation, carefully aspirate the supernatant and transfer it 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.

[0037] (6) Add 500 μl of protein removal solution PR, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid in the collection tube.

[0038] (7) Add 600 μl of wash buffer WB, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid in the collection tube. Repeat the operation once more. After discarding the waste liquid, put it back into the collection tube and centrifuge at 15,000 rpm for 3 min to remove as much wash buffer as possible.

[0039] (8) Remove the adsorption column and transfer it to a 1.5 ml EP tube. Open the cap and let it stand for 3-5 min. Add 40 μl of elution buffer EB to the middle of the adsorption membrane, let it stand at room temperature for 10 min, and centrifuge at 15,000 rpm for 3 min. Determine the concentration of the extracted plasmid.

[0040] 12. Plasmid sequencing: The extracted plasmid was subjected to Sanger sequencing at a concentration of 50 ng / μl. The sequencing results were compared to ensure that the inserted fragment was correct. The plasmid with the correct insertion result was selected for cell liposome transfection experiments.

[0041] The beneficial effects of this invention are as follows: Compared to traditional long-fragment gene editing systems, the novel gene editing system MMEJ-Exo provided by this invention has the following advantages: 1. The efficiency of mediating long-fragment gene editing has been significantly improved; 2. It can stably act on different cell lines, different genome editing sites, and different types of gene editing systems; 3. The MMEJ repair template construction method is simple and can effectively reduce the efficiency of random base changes during gene editing, thereby improving its precision editing efficiency; 4. Enable long-segment editing in non-dividing primary neurons.

[0042] The MMEJ-Exo gene editing system described in this invention provides a simpler, more precise, safer, and more efficient gene editing tool for clinical gene therapy and cell therapy, which is conducive to the development of clinical treatment for gene mutation-related diseases and major diseases such as tumors. Attached Figure Description

[0043] Figure 1 MMEJ-Exo eukaryotic expression vector, in which: (A) is an MS2-sgRNA-spCas9 expression vector, which includes the MS2-sgRNA sequence and the spCas9 sequence, as well as the CBh promoter sequence and the human U6 promoter sequence, which are used to initiate 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 positivity rate reflects the cell transfection efficiency; (B) and (C) are the MCP-RecE expression vector (C) and the MCP-CtIP_N expression vector (B), respectively, which include the EF1A promoter sequence and the MCP-RecE and MCP-CtIP_N sequences; (D) is a schematic diagram of the MMEJ repair template. The MMEJ repair template contains 5-25 bp left and right homologous arms and about 1 kb of target insertion fragment. When the target gene is cut at a specific site, the left and right homologous arms are complementary to the cutting site to achieve precise insertion of the target fragment.

[0044] Figure 2 The MMEJ-Exo gene editing system can effectively improve the efficiency and accuracy of gene editing in HEK293T cells, including: (A) Exonucleases and key functional domains in HEK293T cells HSP90AA1 (A) MMEJ knock-in efficiency at the site; (B) Exonuclease and key functional domains in HEK293T cells HSP90AA1 Site NHEJ editing efficiency; (C) Exonucleases and key functional domains in HEK293T cells HSP90AA1 Precise repair at site 5'; (D) Exonucleases and key functional domains in HEK293T cells HSP90AA1 Precise repair of site 3'.

[0045] Figure 3The MMEJ-Exo gene editing system can effectively improve the efficiency of long-fragment gene editing in multiple eukaryotic cell lines; the MEJ-Exo gene editing system can mediate the precise insertion of exogenous fragments of about 1kb at multiple sites in HEK293T(A), HeLa(B) and MCF7(C) cell lines, with a knock-in efficiency significantly higher than that of the control group; the knock-in efficiency of the MMEJ-CtIP_N system can reach 10~15%.

[0046] Figure 4 The MMEJ-Exo gene editing system enables efficient editing of primary neurons. It can mediate the precise insertion of an approximately 1kb exogenous P2A-mKate fragment into the ACTB murine gene locus in primary mouse neurons, with a significantly higher insertion efficiency than the spCas9-only control group. Detailed Implementation

[0047] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0048] Example 1: Construction of expression vector for the MMEJ-Exo eukaryotic gene editing system This project constructed RecE and CtIP_N eukaryotic expression vectors using the SAM recruitment system. Figure 1 ).

[0049] 1. Figure 1 The construction process of the MS2-sgRNA-spCas9 expression plasmid vector shown in Figure A is as follows: (1) The gene sequences of MS2-sgRNA scaffold and spCas9 were obtained by whole-genome synthesis.

[0050] (2) Construction of the basic plasmid for 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. 6) was digested with restriction endonucleases NdeⅠ (FD0583, ThermoFisher), AgeⅠ (FD1464, ThermoFisher), and HindⅢ (FD0504, ThermoFisher). The reaction was carried out at 37℃ for 40 min. The digestion products were identified by 1.5% agarose gel electrophoresis, and the digestion products were recovered using a gel and PCR clean-up kit (Promega).

[0051] (3) Construction of the MS2-sgRNA-spCas9 gene editing system expression vector: The MS2-sgRNA scaffold (SEQ ID NO.19) and spCas9 gene sequences, along with the enzyme digestion products from step (2), were ligated into the vector plasmid using a Gibson assay at a 1:2 molar ratio. The MS2-sgRNA scaffold sequence was ligated between the NdeI restriction sites, and the spCas9 sequence was ligated between the AgeI and HindIII restriction sites. T5 exonuclease (2×MultiF Seamless Assembly Mix, Abclonal) was mixed with the product at a 1:1 volume ratio. The total volume for the Gibson ligation reaction was 6 μl, and the reaction was carried out at 50℃ for 15 min. The MS2-sgRNA-spCas9 gene editing system expression vector was thus constructed.

[0052] (4) Transformation of competent cells: The competent E. coli cells were thawed on ice. After the competent cells were completely thawed, the Gibson ligation product obtained in step (3) was added to the competent E. coli cells at a volume ratio of 1:10, and the mixture was gently stirred with a pipette tip and left to stand on ice for 10 min. The competent cell mixture was then streaked on LB solid medium containing ampicillin and incubated at 37°C for 12-18 h.

[0053] (5) Single-clone colony amplification and plasmid extraction: After single-clone colonies grow 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 4 ml of bacterial culture and extract plasmids using a high-purity plasmid miniprep kit (Y2204, TIANGEN). Determine the plasmid concentration using a Multiskan SkyHigh full-wavelength microplate reader (A51119700DPC, ThermoScientific).

[0054] (6) Plasmid sequencing: The extracted plasmid was sent to a sequencing company for Sanger sequencing at a concentration of 50 ng / μl. The sequencing results were compared to ensure that the inserted fragment was correct. The plasmid with the correct insertion result was selected for cell transfection experiment. The gene sequence of the expression vector of the MS2-sgRNA-spCas9 gene editing system is shown in SEQ ID NO.7.

[0055] (7) In order to enable the constructed MS2-sgRNA-spCas9 gene editing system expression vector plasmid to effectively target different human gene sites such as ACTB, HSP90AA1, CLTA and SEC61B in various human cell lines such as HEK293T, and to effectively target the mouse gene site of ACTB in mouse primary neurons for gene editing, a 20bp spacer sequence targeting different gene sites was inserted at 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 assay (FL101-01, TransGen Biotech).

[0056] The spacer sequence of the ACTB human gene is shown in SEQ ID NO. 8, the spacer sequence of the HSP90AA1 human gene is shown in SEQ ID NO. 9, the spacer sequence of the CLTA human gene is shown in SEQ ID NO. 10, the spacer sequence of the SEC61B human gene is shown in SEQ ID NO. 11, and the spacer sequence of the ACTB mouse gene is shown in SEQ ID NO. 12.

[0057] 2. Figure 1 The construction procedures for the MCP-RecE and MCP-CtIP_N expression plasmid vectors shown in B and 1C are as follows: (1) The gene sequences of the microbial source Pad RecE shown in SEQ ID NO.1 and the eukaryotic CtIP key functional domain CtIP_N shown in SEQ ID NO.2 were obtained by whole-genome synthesis method.

[0058] (2) The EF1A-MCP expression vector (shown in SEQ ID NO.3) was digested with restriction endonucleases BamHI (FD0054, ThermoFisher) and EcoRI (FD0274, ThermoFisher). The digestion products were collected after identification by 1.5% agarose gel electrophoresis.

[0059] (3) The Pad RecE and CtIP_N gene sequences synthesized in (1) were ligated with the enzyme digestion products in step (2) at a molar ratio of 1:2 using Gibson to construct the MCP-RecE expression vector and the MCP-CtIP_N expression vector, respectively. T5 exonuclease (2×MultiF Seamless Assembly Mix, Abclonal) was mixed with the products at a volume ratio of 1:1. The total volume of the Gibson ligation reaction was 6 μl, and the reaction was carried out at 50 °C for 15 min.

[0060] 3. Figure 1 Construction and amplification of the MMEJ repair template shown in D: The MMEJ repair template is approximately 1 kb and contains approximately 1 kb of P2A-mKate fluorescent protein expression sequence (shown in SEQ ID NO.13) and 5-25 bp homologous arm DNA sequences of ACTB, HSP90AA1, CLTA, SEC61B human genes and ACTB mouse genes on the left and right sides. The MMEJ repair template is amplified by PCR.

[0061] The MMEJ homology repair template sequence of the ACTB human gene is shown in SEQ ID NO.14, the MMEJ homology repair template sequence of the HSP90AA1 human gene is shown in SEQ ID NO.15, the MMEJ homology repair template sequence of the CLTA human gene is shown in SEQ ID NO.16, the MMEJ homology repair template sequence of the SEC61B human gene is shown in SEQ ID NO.17, and the MMEJ homology repair template sequence of the ACTB mouse gene is shown in SEQ ID NO.18.

[0062] Example 2: The MMEJ-Exo gene editing system can effectively improve the efficiency and accuracy of precise gene editing in HEK293T cells. Efficiency evaluation of the MMEJ-Exo gene editing system for precise editing in the HEK293T cell line.

[0063] The specific experimental procedure is as follows: (1) Cell plating: HEK293T human cell line was cultured in DMEM (GS-3301, Homeland Bio) complete medium supplemented with 10% FBS (GL-1001, Homeland Bio) and 1% penicillin / streptomycin (BL505A, biosharp) at 37°C and 5% CO2. 14-16 h before transfection, when the HEK293T cell confluence reached 80-90%, the supernatant was discarded, and the cells were washed once with DPBS (C14190500BT, ThermoFisher), digested with 0.05%-Trypsin-EDTA (25200072, ThermoFisher), transferred to 15 ml centrifuge tubes, and centrifuged at 200 x g for 3 min. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 ml of cell culture medium for cell counting. HEK293T cells were cultured at 7.0-8.0 × 10⁶ cells / mL. 4 Cells were seeded at a density of 60-70% in 48-well plates (11410, Labselect) and transfected when the cell confluence reached 60-70%.

[0064] (2) Cell transfection: Each 48-well cell line was transfected with 25 ng of MMEJ repair template DNA of the human HSP90AA1, DYNLT1, or AAVS1 gene prepared in step 3 of Example 1 and 475 ng of plasmid DNA (including 325 ng of MS2-sgRNA-spCas9 expression vector plasmid prepared in step 1 of Example 1, and 150 ng each of MCP-RecE expression vector plasmid and MCP-CtIP_N expression vector plasmid prepared in step 2 of Example 1). Pre-transfection was performed using Opti-MEM. TM (31985070, ThermoFisher) Dilute P3000 separately TM (L3000015, ThermoFisher) and Lipofectamine 3000 TM (L3000015, ThermoFisher). 48 cells per well, with 12.5 μl Opti-MEM. TM / P3000 TM Dilute 500 ng of DNA with the premixed buffer, then add an equal volume of Opti-MEM. TM Lipofectamine 3000 TM Premix the reagent and let it stand for 15 min. Add 25 μl of the mixed reagent to a single well for transfection, and gently shake the 48-well plate to ensure even distribution in the culture medium. 48 h after transfection, change the cell culture medium. 72 h after transfection, collect the cells and extract genomic DNA using a genomic DNA extraction kit (Qiagen, catalog number #51306).

[0065] (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: 10ul; universal PCR primer (polyT sequence): 2ul; genomic DNA: 1ul; water: 7ul. The reaction program was as follows: 98℃, 5 seconds; 98℃, 10 seconds; 72℃, 30 seconds; (repeat 2 and 3, 25 cycles); 72℃, 10 minutes; store at 4℃.

[0066] After the PCR reaction was completed, the PCR product was purified using a PCR product purification kit (Qiagen, catalog number #28104) and then subjected to next-generation sequencing.

[0067] (4) Next-generation sequencing and data analysis: The above PCR products were prepared for sequencing, the concentration was determined, and 10 ng was added to the Illumina MiSeq Reagent Kit v3 and placed in the Illumina MiSeq sequencer for sequencing. After sequencing, the sequencing results were uploaded to the open-source data analysis tool CRISPResso2 (http: / / crispresso2.pinellolab.org / submission) for data analysis.

[0068] Example 3: The MMEJ-Exo gene editing system can effectively improve the efficiency of long-fragment gene editing in multiple eukaryotic cell lines. P2A-mKate sequences were targeted and inserted into the HSP90AA1, ACTB, CLTA, and SEC61B human gene loci in HEK293T, HeLa, and MCF7 human cell lines using the MMEJ-Exo gene editing system. The editing efficiency was quantitatively evaluated by flow cytometry. The specific experimental procedure is as follows: (1) Cell plating: HEK293T, HeLa, and MCF7 human cell lines were cultured in DMEM (GS-3301, Homeland Bio) complete medium supplemented with 10% FBS (GL-1001, Homeland Bio) and 1% penicillin / streptomycin (BL505A, biosharp) at 37°C and 5% CO2. 14-16 h before transfection, when the cell confluence reached 80-90%, the supernatant was discarded, and the cells were washed once with DPBS (C14190500BT, ThermoFisher), digested with 0.05%-Trypsin-EDTA (25200072, ThermoFisher), transferred to 15 ml centrifuge tubes, and centrifuged at 200 x 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. HEK293T, HeLa, and MCF7 human cell lines were cultured at 7.0–8.0 × 10⁻⁶ cells per cell line. 4 5~6.0×10 4 and 3~4×10 4 Cells were seeded at a density of 60-70% in 48-well plates (11410, Labselect) and transfected when the cell confluence reached 60-70%.

[0069] (2) Cell transfection: Each 48-well cell line was transfected with 25 ng of MMEJ repair template DNA of the human HSP90AA1, DYNLT1, or AAVS1 gene prepared in step 3 of Example 1 and 475 ng of plasmid DNA (including 325 ng of MS2-sgRNA-spCas9 expression vector plasmid prepared in step 1 of Example 1, and 150 ng each of MCP-RecE expression vector plasmid and MCP-CtIP_N expression vector plasmid prepared in step 2 of Example 1). Pre-transfection was performed using Opti-MEM. TM (31985070, ThermoFisher) Dilute P3000 separately TM (L3000015, ThermoFisher) and Lipofectamine 3000 TM (L3000015, ThermoFisher). 48 cells per well, with 12.5 μl Opti-MEM. TM / P3000 TM Dilute 500 ng of DNA with the premixed buffer, then add an equal volume of Opti-MEM. TM Lipofectamine 3000 TMPremix the reagent and let it stand for 15 min. Add 25 μl of the mixed reagent to a single well for transfection, and gently agitate the 48-well plate to ensure even distribution in the culture medium. Replace the cell culture medium 48 h after transfection. Collect cells for analysis 72 h after transfection.

[0070] (3) Flow cytometry analysis: After processing HEK293T, HeLa, and MCF7 human cells in step (2), the MMEJ-Exo long fragment gene editing efficiency was analyzed by flow cytometry. The mKate positivity rate represents the MMEJ-Exo long fragment gene editing efficiency. 72 h after transfection, the supernatant was discarded, the cells were washed once with DPBS, and digested with 0.05%-Trypsin-EDTA. Cells from each well were transferred to the corresponding cell culture wells of a U-shaped 96-well plate and centrifuged at 200xg for 3 min. After centrifugation, the supernatant was discarded, and the cells were resuspended in DPBS containing 2% fetal bovine serum. The results were analyzed using a BD FACSCelesta™ cell analyzer (BD Biosciences).

[0071] Another control group (Ctrl) was set up: a negative control group that was transfected only with MS2-sgRNA-spCas9 and not transfected with MCP-RecE or MCP-CtIP_N.

[0072] Flow cytometry results showed that the MMEJ-Exo gene editing system could mediate the precise insertion of a 1kb exogenous P2A-mKate fragment at the ACTB, HSP90AA1, CLTA, and SEC61B sites in HEK293T, HeLa, and MCF7 human cell lines, with a significantly higher insertion efficiency than the Ctrl control group.

[0073] Example 4: The MMEJ-Exo gene editing system enables highly efficient editing of primary neurons. In primary mouse neurons, approximately 1 kb of P2A-mKate sequence was inserted using the MMEJ-Exo gene editing system at the ACTB murine gene locus. The editing efficiency was evaluated by acquiring images of mKate-positive neurons using fluorescence microscopy. The specific experimental procedure is as follows: (1) Extraction of primary mouse neurons: Cortical and hippocampal neurons were obtained from C57BL / 6J mice at embryonic day 16 (E16). Pregnant mice were anesthetized with isoflurane and the embryos (both male and female) were quickly removed, and the mother mice were euthanized by decapitation. Cortical and hippocampal tissues were then separated and placed in ice bath dissection buffer to maintain their viability, and then digested with 0.25% trypsin-EDTA in an incubator for 15 minutes. After terminating the enzyme reaction with fetal bovine serum, the tissues were gently dissociated into a single-cell suspension by blowing gently with a flame-polished pipette tip. The neurons were centrifuged at low speed (100 g, 5 minutes) and then resuspended in culture medium. Cell density was determined using a cell counter, and the required number of cells (4 × 10⁶ cells per sample) were taken according to experimental needs. 6 (1 cell), then centrifuge again at low speed.

[0074] (2) Electroporation of primary mouse neurons: Each sample was electroporated using 250 ng of ACTB mouse gene MMEJ repair template DNA and 4750 ng of plasmid DNA prepared in step 3 of Example 1 (including 3250 ng of MS2-sgRNA-spCas9 expression vector plasmid prepared in step 1 of Example 1, and 1500 ng of MCP-RecE expression vector plasmid and MCP-CtIP_N expression vector plasmid prepared in step 2 of Example 1). The neurons were resuspended in nuclear transfection solution (Lonza) and 5 μg was added. The cell / DNA suspension was transferred to a dedicated electroporation cuvette and electroporated according to the set program O-005. The electroporated cells were seeded into six-well plates pre-coated with poly-D-lysine (0.05 mg / mL; Sigma-Aldrich) and the medium was replaced with fresh Neurobasal Plus medium to remove cell debris. The primary neurons were cultured for another 10 days, with half the medium being replaced every 4 days. Images of mKate-positive neurons were acquired using a fluorescence microscope on day 10 of culture. The entire culture process was carried out in a humidified incubator at 37 °C and 5% CO2.

[0075] Fluorescence imaging results showed that the MMEJ-Exo gene editing system could mediate the precise insertion of an approximately 1 kb exogenous P2A-mKate fragment into the ACTB murine gene locus in primary mouse neurons, with a significantly higher insertion efficiency than the spCas9-only control group.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the combination of the microbial protein RecE and the key functional domain CtIP_N of the eukaryotic protein CtIP in the construction of a gene editing system, characterized in that... The gene editing system described is mediated by the CRISPR / Cas9 gene editing system in eukaryotic cells, and improves the efficiency of long-fragment gene editing mediated by the gene editing system through the key functional domain CtIP_N of the microbial protein RecE and the eukaryotic protein CtIP. Preferably, the nucleotide sequence of microbial RecE is shown in SEQ ID NO.1, and the nucleotide sequence of the key functional domain CtIP_N of the eukaryotic protein CtIP is shown in SEQ ID NO.

2.

2. A gene editing system based on exonuclease, characterized in that, The gene editing system consists of the following three components: (1) MCP-RecE expression vector and MCP-CtIP_N expression vector; (2) MS2-sgRNA-spCas9 gene editing system expression vector; (3) MMEJ homology repair template.

3. The gene editing system according to claim 2, characterized in that, (1) The MCP-RecE expression vector includes the MCP-RecE sequence, wherein the MCP-RecE sequence is a fusion expression of microbial protein RecE and MCP aptamer protein; the MCP-CtIP_N expression vector includes the MCP-CtIP_N sequence, wherein the MCP-CtIP_N sequence is a fusion expression of eukaryotic protein CtIP key functional domain CtIP_N and MCP aptamer protein; Preferably, the amino acid sequence of the MCP-RecE is shown in SEQ ID NO.4; and the amino acid sequence of the MCP-CtIP_N is shown in SEQ ID NO.

5.

4. The gene editing system according to claim 2, characterized in that, (1) The base vectors of the MCP-RecE expression vector and the MCP-CtIP_N expression vector are plasmids containing MCP aptamer proteins; Preferably, the plasmid containing the MCP aptamer protein is an EF1A-MCP expression vector, and the MCP-RecE expression vector and the MCP-CtIP_N expression vector are constructed by inserting the RecE sequence of the microbial protein or the CtIP_N sequence of the key functional domain of the CtIP of the eukaryotic protein into the BamHI and EcoRI restriction sites of the EF1A-MCP expression vector, respectively. Further preferred, the sequence of the EF1A-MCP expression vector is shown in SEQ ID NO.

3.

5. The gene editing system according to claim 2, characterized in that, (2) The expression vector of the MS2-sgRNA-spCas9 gene editing system includes the MS2-sgRNA scaffold sequence and spCas9 nuclease. Preferably, the expression vector also includes the T2A-EBFP sequence.

6. The gene editing system according to claim 5, characterized in that, The base vector of the MS2-sgRNA-spCas9 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.

6. The MS2-sgRNA-spCas9 gene editing system expression vector is constructed by ligating the MS2-sgRNA scaffold sequence between the NdeI restriction sites of the base vector and ligating the spCas9 nuclease sequence between the AgeI and HindIII restriction sites, and the MS2-sgRNA scaffold sequence is shown in SEQ ID NO.

19.

7. The gene editing system according to claim 5, characterized in that, When the Cas9 nuclease in the MS2-sgRNA-spCas9 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.

7.

8. The gene editing system according to claim 5, characterized in that, The MS2-sgRNA-spCas9 gene editing system expression vector also includes a spacer sequence that targets the gene site to be edited; Preferably, the spacer sequence targeting the gene site to be edited is inserted between the BbsI restriction sites of the MS2-sgRNAscaffold sequence shown in SEQ ID NO.19; Preferably, the gene to be edited is selected from the ACTB human gene, HSP90AA1 human gene, CLTA human gene, SEC61B human gene, and ACTB mouse gene. Further preferred embodiments include the spacer sequence targeting the human ACTB gene as shown in SEQ ID NO. 8, the spacer sequence targeting the human HSP90AA1 gene as shown in SEQ ID NO. 9, the spacer sequence targeting the human CLTA gene as shown in SEQ ID NO. 10, the spacer sequence targeting the human SEC61B gene as shown in SEQ ID NO. 11, and the spacer sequence targeting the mouse ACTB gene as shown in SEQ ID NO.

12.

9. The gene editing system according to claim 2, characterized in that, (3) The MMEJ homology repair template includes a P2A-mKate fluorescent protein expression sequence and a homologous arm DNA sequence of the gene to be edited located to the left and right of the P2A-mKate fluorescent protein expression sequence, as shown in SEQ ID NO.13; Preferably, the DNA sequences of the left and right homologous arms of the gene to be edited are optionally 5-25 bp; Preferably, the gene to be edited is selected from the ACTB human gene, HSP90AA1 human gene, CLTA human gene, SEC61B human gene, and ACTB mouse gene. Further preferred embodiments include the MMEJ homology repair template sequence of the ACTB human gene as shown in SEQ ID NO.14, the MMEJ homology repair template sequence of the HSP90AA1 human gene as shown in SEQ ID NO.15, the MMEJ homology repair template sequence of the CLTA human gene as shown in SEQ ID NO.16, the MMEJ homology repair template sequence of the SEC61B human gene as shown in SEQ ID NO.17, and the MMEJ homology repair template sequence of the ACTB mouse gene as shown in SEQ ID NO.

18.

10. The use of the gene editing system of claim 2 in the preparation of drugs or reagents for diseases related to cell therapy and / or gene therapy; Preferably, the cell therapy is based on one or more of the HEK293T cell line, HeLa cell line, MCF7 cell line, and primary neurons; The gene therapy is based on one or more of the ACTB gene, HSP90AA1 gene, CLTA gene, and SEC61B gene; The application can improve the efficiency of long-fragment gene editing at one or more sites in cells; More preferably, the disease is autosomal dominant polycystic kidney disease (ADPKD), cervical cancer, or breast cancer.

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