DNA polymerase Pol theta mediated long fragment gene editing system

Abstract

The invention relates to application of DNA polymerase in long-fragment precise gene editing and construction in a plurality of human-derived cell lines. The invention belongs to the technical field of biological medicine. A novel gene editing system MMEJ-Pol theta is created by applying DNA polymerase Pol theta and a key functional structure domain, the accuracy, safety and editing efficiency of long-fragment gene editing in a eukaryotic genome are remarkably improved through a micro homologous end linking method, and an effective tool is provided for transformation application of the gene editing system in gene therapy and cell therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. By applying the DNA polymerase Pol θ protein and its key functional domains, a series of safe, precise and efficient gene editing systems for eukaryotic organisms have been successfully constructed, which significantly improves the efficiency of spCas9-mediated long-fragment genome editing and provides new technologies and methods for the translational application of cell therapy for hereditary diseases. Background Technology

[0002] As a novel synthetic biology system, the CRISPR / Cas9 genome editing tool provides strong technical support for genetic engineering, driving the development of basic scientific research and the medical and health industry. Especially for major diseases such as gene mutation-related diseases and cancer, CRISPR / Cas9-based cell therapy strategies are constantly emerging, bringing benefits to patients. In 2023, the UK Medicines and Healthcare products Regulatory Agency (MHRA) and the US Food and Drug Administration (FDA) approved the world's first CRISPR / Cas9 cell therapy product, Casgevy, for the treatment of sickle cell disease, a hereditary blood disorder, marking the beginning of cell therapy's 2.0 era.

[0003] However, existing gene editing methods face several limitations in their translational application in cell therapy. For example, current gene editing strategies mostly involve knocking out target genes rather than precisely repairing or inserting long DNA fragments (greater than 100 bp) into the genome; existing gene editing methods have low efficiency in precisely editing the genome; and the preparation methods for long homologous repair templates that mediate precise editing are complex.

[0004] Therefore, the development of precise, efficient, and simple long-fragment (greater than 100 bp) gene editing tools remains one of the key factors for promoting the further development of cell therapy translational applications. Summary of the Invention

[0005] This patent describes a series of novel gene editing systems constructed using a DNA polymerase, Pol θ, and its key functional domains, significantly improving the efficiency of spCas9-mediated long-fragment genome editing. By applying Pol θ to the spCas9 editing system, a DNA polymerase θ editor (hereinafter referred to as PET) was developed. Based on PET, the polymerase domain (POL domain) and helicase-like domain (HEL domain) of Pol θ were engineered to develop a polymerase domain editing system (hereinafter referred to as pPET) and a helicase-like domain editing system (hereinafter referred to as hPET). These gene editing systems overcome the technical challenge of low efficiency in long-fragment editing using traditional tools, providing new tools for the development of in vivo and in vitro cell therapy.

[0006] The purpose of this invention is to provide a series of novel long-fragment gene editing systems that enable precise, efficient, and simple long-fragment gene editing operations on eukaryotic genomes, and to confirm their gene editing capabilities in cell lines from different species. This provides an effective application tool for cell therapy of gene mutation-related diseases and promotes the development of translational applications in clinical treatment.

[0007] The technical solution of this invention is as follows: The first objective of this invention is to provide the application of DNA polymerase Pol θ and its functional domains in the construction of gene editing systems, wherein the functional domains are HEL domain and POL domain, and the DNA polymerase Pol θ and its functional domains can improve the efficiency of long-fragment gene editing mediated by the CRISPR / Cas9 gene editing system in eukaryotic cells. Preferably, the nucleotide sequences of DNA polymerase Pol θ, HEL domain, and POL domain are as shown in SEQ ID NO. 8, 9, and 10.

[0008] The Pol θ protein described in this invention can improve the efficiency of long-fragment precise gene editing mediated by the CRISPR / Cas9 gene editing system. The Pol θ protein described in this invention participates in the construction of the PET editing system. hPET and pPET are engineered based on the PET editing system, applying the Pol θ functional domain to further improve the editing efficiency of long-fragment (nearly kilobase level) precise gene editing. The pPET editing system achieves three times the precision editing efficiency of traditional spCas9.

[0009] The second objective of this invention is to provide a CRISPR / Cas9 gene editing system based on the DNA polymerase Pol θ and its functional domains, wherein the gene editing system comprises the following three components: (1) MCP-POL θ expression vector or MCP-HEL expression vector or MCP-POL expression vector; (2) MS2-sgRNA-Cas9 gene editing system expression vector; (3) MMEJ homology repair template.

[0010] Furthermore, the PET, hPET, and pPET editing systems insert nucleotide sequences of DNA polymerase Pol θ, HEL domain, and POL domain after the MCP sequence in the EF1A-MCP expression vector, respectively.

[0011] Furthermore, (1) the MCP-Pol θ expression vector, MCP-HEL expression vector, and MCP-POL expression vector include the MCP-Pol θ, MCP-HEL, and MCP-POL sequences, wherein the MCP-Pol θ, MCP-HEL, and MCP-POL sequences are expressed by fusing DNA polymerase Pol θ, its functional domains HEL domain and POL domain with MCP aptamer protein, respectively; the nucleotide sequences of DNA polymerase Pol θ, HEL domain, and POL domain are shown in SEQ ID NO. 8, 9, and 10. Preferably, the base vector for the MCP-Pol θ expression vector, MCP-HEL expression vector, and MCP-POL expression vector is the EF1A-MCP expression vector. The nucleotide sequences of the DNA polymerase Pol θ, HEL domain, and POL domain are respectively inserted between the BamHI and EcoRI restriction sites of the EF1A-MCP expression vector to construct the vector. Further preferred, the sequence of the EF1A-MCP expression vector is shown in SEQ ID NO.21.

[0012] Furthermore, (1) the amino acid sequences of the DNA polymerase Pol θ, HEL domain, and POL domain are shown in SEQ ID NO.11, 12, and 13.

[0013] Furthermore, the MS2-sgRNA-Cas9 gene editing system expression vector includes an MS2-sgRNA sequence and a Cas9 nuclease, wherein the MS2-sgRNA sequence includes an MS2-sgRNA scaffold sequence; preferably, the expression vector also contains a T2A-EBFP sequence.

[0014] Furthermore, (2) 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.22; the basic vector includes the T2A-EBFP sequence, which is used to evaluate cell transfection efficiency.

[0015] The MS2-sgRNA-Cas9 gene editing system expression vector was constructed by inserting the MS2-sgRNA scaffold sequence between the NdeI restriction sites of the pU6-CBh-T2A-EBFP basic vector, and the Cas9 nuclease sequence between the AgeI and HindIII restriction sites.

[0016] Furthermore, (2) the Cas9 nuclease in the expression vector of the MS2-sgRNA-Cas9 gene editing system is spCas9.

[0017] Furthermore, (2) the MS2-sgRNA-Cas9 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 inserted between the BbsⅠ restriction sites of the MS2-sgRNA scaffold sequence in the MS2-sgRNA-Cas9 gene editing system expression vector of claim 6 or 7; Preferably, the gene to be edited is selected from the human gene HSP90AA1, the human gene ACTB, the human gene RAB11A, the human gene SEC61B, the mouse gene ACTB, or the mouse gene TUBB3.

[0018] Preferably, the spcaer sequence of the HSP90AA1 human gene is shown in SEQ ID NO.2, the spcaer sequence of the ACTB human gene is shown in SEQ ID NO.3, the spcaer sequence of the RAB11A human gene is shown in SEQ ID NO.4, the spcaer sequence of the SEC61B human gene is shown in SEQ ID NO.5, the spcaer sequence of the ACTB mouse gene is shown in SEQ ID NO.6, and the spcaer sequence of the TUBB3 mouse gene is shown in SEQ ID NO.7.

[0019] Based on the working principle of the SAM recruitment system, the MCP aptamer of the MCP-Pol θ expression vector specifically binds to the MS2 stem-loop structure of the MS2-sgRNA-Cas9 gene editing system expression vector, enabling Pol θ and sgRNA-Cas9 to synergistically perform gene editing functions in eukaryotic cells.

[0020] Furthermore, (3) the MMEJ homology repair template includes the sequence to be inserted and the DNA sequence of the homologous arm of the gene to be edited located to the left and right of the sequence to be inserted.

[0021] In a particular embodiment, the sequence to be inserted is a P2A-mKate fluorescent protein expression sequence, as shown in SEQ ID NO.14 (780 bp).

[0022] Preferably, the gene to be edited is selected from at least one of the following: HSP90AA1 human gene, ACTB human gene, RAB11A human gene, SEC61B human gene, ACTB mouse gene, and TUBB3 mouse gene. Further preferred, the DNA sequences of the left and right homologous arms of the gene to be edited are both 25 bp; Further preferably, the micro-homology repair template sequence of the HSP90AA1 human gene is shown in SEQ ID NO.15, the micro-homology repair template sequence of the ACTB human gene is shown in SEQ ID NO.16, the micro-homology repair template sequence of the RAB11A human gene is shown in SEQ ID NO.17, the micro-homology repair template sequence of the SEC61B human gene is shown in SEQ ID NO.18, the micro-homology repair template sequence of the ACTB mouse gene is shown in SEQ ID NO.19, and the micro-homology repair template sequence of the TUBB3 mouse gene is shown in SEQ ID NO.20.

[0023] 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 HEK293T cells, MCF7, or N2A cell lines. Preferably, the gene therapy is to treat the disease by editing one or more of the following: HSP90AA1 human gene, ACTB human gene, RAB11A human gene, SEC61B human gene, ACTB mouse gene, and TUBB3 mouse gene. Preferably, the application can improve the efficiency of long-fragment gene editing at multiple sites in cells.

[0024] A fourth objective of this invention is to provide the application of the aforementioned gene editing system in improving the efficiency of editing long fragments at multiple sites of one or more of the following human genes: HSP90AA1, ACTB, RAB11A, SEC61B, ACTB, and TUBB3.

[0025] The beneficial effects of this invention are as follows: Compared to traditional gene editing systems, the novel gene editing systems PET, pPET, and hPET provided by this invention have the following advantages: 1) The efficiency of mediating long fragment (greater than 100 bp) gene editing was significantly improved; 2) It can stably act on different cell lines, different genome editing sites, and different types of gene editing systems.

[0026] This type of gene editing system provides a safe, precise, and efficient gene editing tool for clinical cell therapy, which helps to promote the rapid development of precision medicine. Attached Figure Description

[0027] Figure 1 Construction diagrams of plasmid vectors for PET, hPET, and pPET editing systems, where: A is a schematic diagram of the MS2-sgRNA-spCas9 plasmid vector, showing the basic backbone structure of the vector and the arrangement of its main functional elements, including the promoter region, coding sequence region, and terminator region; B shows the construction diagram of the MCP-Pol θ fusion protein expression vector, which contains a fusion protein expression cassette of MCP protein driven by the EF1A promoter and DNA polymerase θ. Pol θ has microhomological end-mediated repair activity. C shows the construction diagram of the MCP-POL domain fusion protein expression vector. The EF1A promoter-driven expression of the MCP protein and POL domain fusion protein is used to enhance DNA repair and recombination capabilities. D is a diagram of the construction of the MCP-HEL domain fusion protein expression vector. The expression of the fusion protein of MCP protein and HEL domain driven by the EF1A promoter helps the DNA double-strand unwinding and repair process.

[0028] Figure 2 SAM recruitment system and micro-homogeneity repair template design diagram, in which: A is a schematic diagram of the PET, pPET, and hPET editing systems for editing target genes. The SAM recruitment system mediates the Cas9 to generate DNA double bond breaks at the target site, thereby inserting the target gene sequence. B is a schematic diagram of the MMEJ repair template, which contains P2A-mKate fluorescent protein (780 bp) and HA microhomologous arms (25 bp + 25 bp). C represents the MMEJ repair template sequence, the bold text represents the mKate fluorescent protein sequence, and the unbold text represents the left and right 25 bp homologous arm sequences; D is a schematic diagram of the microhomology repair mechanism, showing the end-joining repair process mediated by microhomology sequences after DNA double-strand breaks, including steps such as 5'-3' exonuclease treatment, microhomology sequence annealing, and DNA ligation.

[0029] Figure 3 The editing efficiency of PET, pPET, and hPET systems at different gene loci in HEK293T cells was validated, including: A, B, C, and D represent the mKate knock-in efficiency at human HSP90AA1, ACTB, RAB11A, and SEC61B gene loci. The knock-in efficiency of Pol θ and its functional domains was significantly higher than that of the NTC and spCas9-only control groups, with pPET showing the best editing efficiency. E is the editing efficiency fluorescence analysis diagram, comparing the editing efficiency differences at the human HSP90AA1 site between the three systems PET, pPET, and hPET and the spCas9 ctrl group.

[0030] Figure 4 PET, pPET, and hPET gene editing systems can effectively improve the efficiency of long-fragment gene editing in multiple cell lines, among which: The PET, pPET, and hPET gene editing systems can precisely insert 830bp exogenous fragments at multiple sites in MCF7 (A, B) and N2A cell lines (C, D), with significantly higher knock-in efficiency than the control group. Flow cytometry analysis showed that the pPET and hPET gene editing systems were significantly more efficient than the spCas9 editing system, with a 2-3 fold increase in efficiency. NTC: control group without sgRNA; Cas9Control: control group without Pol θ. Detailed Implementation

[0031] The precise, safe, and efficient gene editing mediated by the PET, pPET, and hPET gene editing systems of this invention is mainly achieved through the following steps: (1) Cell culture: HEK293T and other cell lines were cultured using Dulbecco modified Eagle medium (DMEM) high-glucose medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin and 100 μg / ml streptomycin, in a 5% CO2, 37°C constant temperature cell culture incubator.

[0032] Cell resuscitation: Remove the cells to be resuscitated from liquid nitrogen and quickly place them in 37°C warm water to thaw. After complete thawing, transfer 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, 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.

[0033] Cell passage: When 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 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.

[0034] Cell cryopreservation: Once cell confluence reaches 95%, discard the culture medium supernatant, wash once with DPBS, digest 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 HEK293T cells to a 2ml cryovial and incubate overnight at -80℃, then transfer to liquid nitrogen for storage.

[0035] (2) 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 TMPremix the solution and let it stand for 15 minutes. Add 25 μl of the mixed transfection reagent to each well for transfection, and gently shake the 48-well plate to ensure even distribution in the culture medium. Replace the cell culture medium 48 hours after transfection. Collect cells 72 hours after transfection for subsequent experimental analysis.

[0036] (3) Flow cytometry analysis: The mKate fluorescent protein insertion efficiency 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.

[0037] The key components of the PET, pPET, and hPET gene editing systems, namely the MCP-Polθ, MCP-POL, MCP-HEL, and MS2-sgRNA-spCas9 expression vectors, are mainly achieved through the following steps: (4) Plasmid construction: The gene sequences of Pol θ, POL domain, HEL domain, and MS2-sgRNA-spCas9 were obtained through whole-genome synthesis. Pol θ, POL domain, and HEL domain were ligated into the EF1A-MCP expression vector plasmid using a Gibson assay, or MS2-sgRNA-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.

[0038] (5) 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.

[0039] (6) 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 seconds, discard the supernatant, and collect the bacterial precipitate.

[0040] 2. Add 250 μl of solution P1 to resuspend the bacterial precipitate, and vortex until completely suspended.

[0041] 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.

[0042] 4. Add 350 μl of solution P3 and immediately gently invert the container 6 times. After a white flocculent precipitate appears, centrifuge at 12,000 rpm for 10 min.

[0043] 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.

[0044] 6. Add 500 μl of protein removal solution PR, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube.

[0045] 7. Add 600 μl of wash buffer (WB), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube. Repeat the operation once more. After discarding the waste liquid, return the tube to the collection tube and centrifuge at 15,000 rpm for 3 minutes to remove as much wash buffer as possible.

[0046] 8. Transfer the adsorption column 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.

[0047] (7) Plasmid sequencing: The extracted plasmid was subjected to Sanger sequencing at a concentration of 100 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.

[0048] Example 1: Construction of expression vectors for PET, pPET, and hPET gene editing systems This project constructs eukaryotic expression vectors for Pol θ and its functional domains using the SAM recruitment system. Figure 1 ).

[0049] 1. Figure 1 The construction process of the expression plasmid vector for MS2-sgRNA-spCas9 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. 1) 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 and spCas9 gene sequences and 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. Gibson enzyme (2×MultiF Seamless Assembly Mix, Abclonal) was mixed with the products at a 1:1 volume ratio. The total volume of 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 100 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.1.

[0055] (7) In order to enable the constructed MS2-sgRNA-spCas9 gene editing system expression vector plasmid to effectively target different gene sites of HSP90AA1, ACTB, RAB11A, SEC61B and TUBB3 genes in various eukaryotic cell lines such as HEK293T, a 20 bp 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 gene editing system expression vector plasmid using the Golden Gate assay (FL101-01, TransGen Biotech).

[0056] The specific spcaer sequences of the HSP90AA1 gene are shown in SEQ ID NO.2, the ACTB gene in SEQ ID NO.3, the RAB11A gene in SEQ ID NO.4, the SEC61B gene in SEQ ID NO.5, the murine ACTB gene in SEQ ID NO.6, and the murine TUBB3 gene in SEQ ID NO.7.

[0057] 2. Figure 1 The construction process of the EF1A-MCP-Pol θ expression vector shown in Figure B is as follows: (1) The amino acid sequence of DNA polymerase Pol θ shown in SEQ ID NO.11 was obtained by whole-gene synthesis method.

[0058] (2) The EF1A-MCP expression vector (shown in SEQ ID NO.21) 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 DNA polymerase Pol θ gene sequence synthesized in (1) was ligated with the enzyme digestion product in step (2) at a molar ratio of 1:2 using Gibson. The Gibson enzyme (2×MultiF Seamless Assembly Mix, Abclonal) was mixed with the product 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 The construction process of the EF1A-MCP-HEL domain expression plasmid vector shown in C is as follows: (1) The amino acid sequence of the HEL domain shown in SEQ ID NO.12 was obtained by whole-genome synthesis.

[0061] (2) The EF1A-MCP expression vector (shown in SEQ ID NO.21) 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.

[0062] (3) The HEL domain nucleotide sequence synthesized in (1) was ligated with the enzyme digestion product in step (2) at a molar ratio of 1:2 using Gibson. The Gibson enzyme (2×MultiF Seamless Assembly Mix, Abclonal) was mixed with the product 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.

[0063] 4. Figure 1 The construction process of the EF1A-MCP-POL domain expression plasmid vector shown in Figure D is as follows: (1) The amino acid sequence of the POL domain shown in SEQ ID NO.13 was obtained by whole-genome synthesis.

[0064] (2) The EF1A-MCP expression vector (shown in SEQ ID NO.21) 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.

[0065] (3) The POL domain nucleotide sequence synthesized in (1) was ligated with the enzyme digestion product in step (2) at a molar ratio of 1:2 using Gibson. The Gibson enzyme (2×MultiF Seamless Assembly Mix, Abclonal) was mixed with the product 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.

[0066] Example 2: Construction and amplification of micro-homology repair templates Design a micro-homogeneous repair template ( Figure 2 The template contains a 780 bp P2A-mKate fluorescent protein expression sequence (shown in SEQ ID NO. 14) and 20 bp homologous arms on each side when targeting HSP90AA1, ACTB, RAB11A, SEC61B, and TUBB3 genes. The microhomology repair template is amplified by PCR. The microhomology repair template sequences for the human HSP90AA1 gene are shown in SEQ ID NO. 15, the human ACTB gene in SEQ ID NO. 16, the human RAB11A gene in SEQ ID NO. 17, the human SEC61B gene in SEQ ID NO. 18, the murine ACTB gene in SEQ ID NO. 19, and the murine TUBB3 gene in SEQ ID NO. 20.

[0067] Example 3: PET, pPET, and hPET gene editing systems can effectively improve the efficiency and accuracy of precise gene editing in HEK293T cells. Efficiency evaluation of precise editing using PET, pPET, and hPET gene editing systems in the HEK293T cell line.

[0068] 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%.

[0069] (2) Cell transfection: Each 48-well cell line was transfected with 10 ng of MMEJ repair template DNA for the HSP90AA1, ACTB, RAB11A, or SEC61B genes 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, 140 ng each of the EF1A-MCP-Polθ expression vector plasmid prepared in step 2 of Example 1, the EF1A-MCP-HEL expression vector plasmid prepared in step 3, and the EF1A-MCP-POL expression vector plasmid prepared in step 4). Opti-MEM was used beforehand. 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 10 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 flow cytometry analysis 72 h after transfection.

[0070] (3) Flow cytometry analysis: After treatment with step (2) on HEK293T cells, the efficiency of MMEJ-mediated precise editing of long-fragment genes such as PET, pPET, and hPET was analyzed by flow cytometry. The mKate positivity rate represents the efficiency of precise editing of long-fragment genes such as PET, pPET, and hPET. 72 h after transfection, the supernatant was discarded, and the cells were washed once with DPBS and digested with 0.05%-Trypsin-EDTA. Cells in each well were transferred to 1.5 ml EP tubes 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. The results were analyzed using a BD FACSCelesta™ cell analyzer (BD Biosciences).

[0071] (4) Fluorescence microscopy analysis: After HEK293T cells were treated in step (2) for 72 hours, mKate-positive cells in each transfection group were photographed using an inverted fluorescence microscope (YHF40, Shanghai Yuehe). The mKate positivity rate represents the efficiency of precise editing of long fragment genes in PET, pPET, and hPET. Control groups were set up as follows: NTC: negative control group without gene site targeting sgRNA; Cas9 control: negative control group only transfected with MS2-sgRNA-spCas9, without transfection with MCP-POLθ.

[0072] The results showed that, compared with the NTC and Cas9 control groups, the PET, pPET, and hPET groups effectively mediated the precise insertion of the exogenous P2A-mKate gene sequence into the HSP90AA1, ACTB, RAB11A, and SEC61B gene loci in the HEK293T human cell genome. Fluorescence microscopy results indicated that PET, pPET, and hPET all effectively mediated higher insertion efficiency of the P2A-mKate gene sequence into the HSP90AA1 gene locus. Figure 3 E).

[0073] Example 4: pPET and hPET gene editing systems 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 (MCF7), and ACTB, TUBB3 (N2A) gene loci in MCF7 and N2A cell lines using the pPET and hPET gene editing systems. The editing efficiency was quantitatively evaluated by flow cytometry. The specific experimental procedure is as follows: (1) Cell plating: MCF7 and N2A 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.

[0074] (2) Cell transfection: Each 48-well cell line was transfected with 10 ng of MMEJ repair template DNA of the human HSP90AA1, ACTB, or TUBB3 gene prepared in Example 2 and 490 ng of plasmid DNA (including 350 ng of MS2-sgRNA-spCas9 expression vector plasmid prepared in step 1 of Example 1 and 140 ng of MCP-POL / HEL expression vector plasmid prepared in step 2 of Example 1). Pre-treatment with Opti-MEM was performed. 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 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.

[0075] (3) Flow cytometry analysis: After treatment in step (2) on MCF7 and N2A cells, the efficiency of MMEJ-mediated precise editing of long-fragment genes in pPET and hPET was analyzed by flow cytometry. The mKate positivity rate represents the efficiency of precise editing of long-fragment genes in pPET and hPET. 72 h after transfection, the supernatant was discarded, and 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).

[0076] The control groups were set up as follows: Blank: blank control group without transfection DNA; NTC: negative control group without gene site targeting sgRNA; Cas9 Control: negative control group only transfected with MS2-sgRNA-spCas9, without transfection with MCP-POL / HEL.

[0077] Flow cytometry results showed that the pPET and hPET gene editing systems could edit the HSP90AA1 gene locus in the MCF7 cell line ( Figure 4 B left), ACTB human gene locus ( Figure 4 B right) and the ACTB gene locus of the N2A cell line ( Figure 4 D left), TUBB3 gene locus ( Figure 4 The D-right) mediates the precise insertion of a 780bp exogenous P2A-mKate fragment, with a significantly higher insertion efficiency than the Cas9Control control group.

[0078] 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 DNA polymerase Pol θ and its functional domains in constructing gene editing systems, characterized in that, The functional domains of the DNA polymerase Pol θ are HEL domain and POL domain. The DNA polymerase Pol θ and its functional domains can improve the efficiency of long-fragment gene editing mediated by the CRISPR / Cas9 gene editing system in eukaryotic cells. Preferably, the nucleotide sequences of the DNA polymerase Pol θ, HEL domain and POL domain are as shown in SEQ ID NO. 8, 9 and 10.

2. A CRISPR / Cas9 gene editing system based on DNA polymerase Pol θ and its functional domains, characterized in that, The gene editing system consists of the following three components: (1) MCP-Polθ expression vector or MCP-HEL expression vector or MCP-POL expression vector; (2) MS2-sgRNA-Cas9 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-Pol θ expression vector, MCP-HEL expression vector, and MCP-POL expression vector include the MCP-Pol θ, MCP-HEL, and MCP-POL sequences, wherein the MCP-Pol θ, MCP-HEL, and MCP-POL sequences are expressed by fusing the DNA polymerase Pol θ, HEL domain, and POL domain with the MCP aptamer protein, respectively; the nucleotide sequences of the DNA polymerase Pol θ, HEL domain, and POL domain are shown in SEQ ID NO. 8, 9, and 10, respectively. Preferably, the base vector for the MCP-Pol θ expression vector, MCP-HEL expression vector, and MCP-POL expression vector is the EF1A-MCP expression vector; the nucleotide sequences of the DNA polymerase Pol θ, HEL domain, and POL domain are respectively inserted between the BamHI and EcoRI restriction sites of the EF1A-MCP expression vector to construct the vector. Further preferred, the sequence of the EF1A-MCP expression vector is shown in SEQ ID NO.

21.

4. The gene editing system according to claim 2, characterized in that, (1) The amino acid sequences of the DNA polymerase Pol θ, HEL domain and POL domain are shown in SEQ ID NO.11, 12 and 13.

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. Preferably, the expression vector also contains a T2A-EBFP sequence.

6. The gene editing system according to claim 2, characterized in that, (2) The basic vector of the MS2-sgRNA-Cas9 gene editing system expression vector is pU6-CBh-T2A-EBFP expression vector, and the nucleotide sequence of pU6-CBh-T2A-EBFP expression vector is shown in SEQ ID NO.22; The MS2-sgRNA-Cas9 gene editing system expression vector is constructed by ligating the MS2-sgRNA scaffold sequence between the NdeI restriction sites of the basic vector and ligating the Cas9 nuclease sequence between the AgeI and HindIII restriction sites. Preferably, the nucleotide sequence of the MS2-sgRNA-Cas9 gene editing system expression vector is shown in SEQ ID NO.

1.

7. The gene editing system according to claim 2, characterized in that, (2) The Cas9 nuclease in the expression vector of the MS2-sgRNA-Cas9 gene editing system is spCas9.

8. The gene editing system according to claim 2, characterized in that, (2) The MS2-sgRNA-Cas9 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 inserted between the BbsⅠ restriction sites of the MS2-sgRNA scaffold sequence in the aforementioned MS2-sgRNA-Cas9 gene editing system expression vector; Preferably, the gene to be edited is selected from the HSP90AA1 human gene, the ACTB human gene, the RAB11A human gene, the SEC61B human gene, the ACTB mouse gene, or the TUBB3 mouse gene. Preferably, the spcaer sequence of the HSP90AA1 human gene is shown in SEQ ID NO.2, the spcaer sequence of the ACTB human gene is shown in SEQ ID NO.3, the spcaer sequence of the RAB11A human gene is shown in SEQ ID NO.4, the spcaer sequence of the SEC61B human gene is shown in SEQ ID NO.5, the spcaer sequence of the ACTB mouse gene is shown in SEQ ID NO.6, and the spcaer sequence of the TUBB3 mouse gene is shown in SEQ ID NO.

7.

9. The gene editing system according to claim 2, characterized in that, (3) The MMEJ homology repair template includes the sequence to be inserted and the DNA sequences of the homologous arms of the gene to be edited located to the left and right of the sequence to be inserted; Preferably, the sequence to be inserted is a P2A-mKate fluorescent protein expression sequence, as shown in SEQ ID NO.14; Preferably, the gene to be edited is selected from at least one of the following: HSP90AA1 human gene, ACTB human gene, RAB11A human gene, SEC61B human gene, ACTB mouse gene, and TUBB3 mouse gene. Further preferred, the DNA sequences of the left and right homologous arms of the gene to be edited are both 25 bp; Further preferably, the micro-homology repair template sequence of the HSP90AA1 human gene is shown in SEQ ID NO.15, the micro-homology repair template sequence of the ACTB human gene is shown in SEQ ID NO.16, the micro-homology repair template sequence of the RAB11A human gene is shown in SEQ ID NO.17, the micro-homology repair template sequence of the SEC61B human gene is shown in SEQ ID NO.18, the micro-homology repair template sequence of the ACTB mouse gene is shown in SEQ ID NO.19, and the micro-homology repair template sequence of the TUBB3 mouse gene is shown in SEQ ID NO.

20.

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 HEK293T cells, MCF7, or N2A cell lines. Preferably, the gene therapy is to treat the disease by editing at least one of the following: HSP90AA1 human gene, ACTB human gene, RAB11A human gene, SEC61B human gene, ACTB mouse gene, and TUBB3 mouse gene. Preferably, the application can improve the efficiency of long-fragment gene editing at multiple sites in cells.

11. The application of the gene editing system of claim 2 in improving the efficiency of long fragment gene editing at multiple sites of at least one of the following: HSP90AA1 human gene, ACTB human gene, RAB11A human gene, SEC61B human gene, ACTB mouse gene, and TUBB3 mouse gene.

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