A method for site-specific integration of a gene of interest into a specific site of an immune cell and uses thereof

Abstract

The application discloses a method for site-specific integration of a target gene into a specific site of an immune cell and application thereof, in particular, a method for site-specific integration of a target gene (for example, a CAR gene) into a specific site (for example, HPK1) of an immune cell based on a non-viral vector and application thereof. The method can precisely integrate the target gene (for example, a CAR gene) into the specific site (for example, HPK1) of the immune cell, realize one-step completion of gene knockout and target gene introduction, and prepare a cell with a specific site of gene knockout and stable expression of the target gene. The method has the characteristics of simple preparation, low cost, stable expression, stable function and no influence on the gene characteristics of the cell. The obtained cell has the characteristics of high killing efficiency and strong infiltration capacity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for targeted integration of a target gene (e.g., CAR gene) into a specific site (e.g., HPK1) on immune cells using a non-viral vector, and its application. Background Technology

[0002] Gene therapy refers to the introduction of exogenous genes into target cells to correct or compensate for diseases caused by defective or abnormal genes, thereby achieving therapeutic goals. Gene therapy is mainly used to treat diseases that pose a serious threat to human health, including: malignant tumors, genetic diseases (such as hemophilia, congenital amaurosis, and cystic fibrosis), cardiovascular diseases, infectious diseases (such as AIDS), and autoimmune diseases (such as rheumatoid arthritis, ankylosing spondylitis, and psoriasis).

[0003] Cancer is one of the major diseases threatening human health, and in recent years, immunotherapy for cancer has gradually become a research hotspot in cancer treatment. Cancer immunotherapy involves modifying, culturing, and expanding immune cells collected from the human body in vitro, and then reinfusing them into the patient to stimulate and enhance the body's own immune function, thereby inhibiting tumor growth and killing tumor cells.

[0004] Chimeric antigen receptor T-cell (CAR-T cell) immunotherapy has achieved significant success in the treatment of hematologic malignancies. CAR-carrying T cells can directly recognize target antigens, regardless of whether the antigen is presented on the MHC, thus avoiding MHC restriction. Upon stimulation by the target antigen in vivo, it induces a series of cellular immune responses, ultimately killing the target cells. Traditional CAR-T therapy utilizes genetic engineering technology to transfer CARs into T cells via viral vectors. This strategy often requires viral vectors such as lentiviruses for infection. On the one hand, viral vector production is costly and relatively complex; on the other hand, it introduces safety risks related to random insertion, such as insertion point mutations and the induction of cell transformation or even carcinogenesis.

[0005] Based on this, some site-specific integration techniques have been disclosed in the prior art. For example, patent application CN105524943A discloses a method for site-specific integration of CAR genes into the AAVS1 site of T cells based on double-stranded microcarriers, and patent application CN111944848A discloses a method for improving site-specific gene integration into cells based on electroporation and CRISPER-Cas9 technology. Patent application CN113005092A discloses the preparation of PD1-knockout CAR-T cells targeting LMP1, using LMP1-targeting CAR-T cells in combination with PD1 knockout therapy. However, the methods in the prior art often suffer from drawbacks such as complex procedures and low CAR transduction efficiency. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a method for targeted integration of a target gene (e.g., CAR gene) into a specific site (e.g., HPK1) of immune cells using a non-viral vector. This method can achieve the knockout of a specific gene site and the introduction of the target gene in one step, simplifying the process, reducing production costs, and shortening the preparation time.

[0007] In a first aspect of the present invention, a method for site-specific integration of a target gene into a specific site on an immune cell is provided, comprising the following steps:

[0008] S1, constructing a homologous recombination repair template vector;

[0009] S2, the gene editing system and the vector obtained in step S1 are introduced into immune cells;

[0010] Optionally, in step S3, the immune cells obtained in step S2 are cultured and identified.

[0011] In step S1, the vector is selected from adeno-associated virus (AAV), minicircle DNA (mcDNA), double-stranded DNA (dsDNA), or single-stranded DNA (ssDNA), especially minicircle DNA.

[0012] Specifically, the homologous recombination repair template in step S1 includes a homologous arm and a target gene. More specifically, the homologous recombination repair template includes, from 5' to 3', the following sequence: target sequence (TSF), left homologous arm (LHA), promoter, target gene, polyA, right homologous arm (RHA), and target sequence (TSF).

[0013] Specifically, the target gene can be a CAR gene; more specifically, the targets recognized by CAR can be: ROR1, Her2, Ll-CAM, CD19, CD20, CD22, CEA, hepatitis B surface antigen, folate receptor antibody, CD23, CD24, CD30, CD33, CD38, CD276, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kdr, κ light chain, Lewis Y, L1 cell adhesion molecule (CD171), MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, tumor-fetal antigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, MAGEA3, CE7, Wilms tumor 1 (WT-1), cyclin A1 (CCNA1), BCMA, and interleukin 12; in some embodiments of the present invention, the target of CAR recognition is CD19, i.e., CD19 CAR.

[0014] In one embodiment of the invention, the CAR gene comprises, and in particular, is composed of, the nucleotide sequence shown in SEQ ID NO:17.

[0015] Specifically, the specific sites can be HPK1, PD-1, TRAC, especially HPK1, and especially HPK1 EXON.

[0016] Specifically, the TSF contains a nucleotide sequence as shown in SEQ ID NO: 16, or the sequence of the TSF is as shown in SEQ ID NO: 16.

[0017] Specifically, the lengths of the left and right homologous arms are 200-1000 bp (e.g., 200, 300, 400, 500, 600, 700, 800, 900, 1000 bp). In one embodiment of the present invention, the left and right homologous arms respectively contain nucleotide sequences as shown in SEQ ID NO: 9 and 10, or as shown in SEQ ID NO: 9 and 10, respectively. In another embodiment of the present invention, the left and right homologous arms respectively contain nucleotide sequences as shown in SEQ ID NO: 11 and 12, or as shown in SEQ ID NO: 11 and 12, respectively. In another embodiment of the present invention, the left and right homologous arms respectively contain nucleotide sequences as shown in SEQ ID NO: 13 and 14, or as shown in SEQ ID NO: 13 and 14, respectively.

[0018] Specifically, the promoter can be EF1αpromoter, whose nucleotide sequence is shown in SEQ ID NO: 15.

[0019] Specifically, polyA can be BGHpA, SV40 polyA, or WPRE, and their nucleotide sequences are shown in SEQ ID NO: 6-8, respectively; in one embodiment of the present invention, polyA is SV40polyA.

[0020] Specifically, the gene editing system in step S2 is a gene editing system that reduces the expression of a specific site (e.g., HPK1) in immune cells. It can be a CRISPR system, ZFN, TALEN, etc., especially a CRISPR system, such as the CRISPR / Cas9 system, CRISPR / Cas12a system, CRISPR / Cas13 system, especially the CRISPR / Cas9 system.

[0021] Specifically, the CRISPR system includes gRNA and nucleases, or is composed of them.

[0022] Specifically, the nuclease can be SpCas9, SaCas9, eSpCas9, Cas12a, Cas13, or cpf1; in some embodiments of the present invention, the nuclease can be SpCas9.

[0023] Specifically, the gRNA specifically targets a specific site (e.g., HPK1), which may have a targeting domain complementary to the target sequence; in some embodiments of the invention, the target sequence comprises, or is composed of, any of the nucleotide sequences shown in SEQ ID NO: 1-5.

[0024] In some embodiments of the present invention, the gRNA comprises, and in particular, consists of, the nucleotide sequence shown in SEQ ID NO:18.

[0025] Specifically, the gRNA also includes chemical modifications of the bases, such as methylation or thioation or a combination of both; more specifically, the 5' end and / or the 3' end of the gRNA are modified with 2'-O-methylation and / or 3'-thiosulfate, with each of the three bases containing methylation; in some embodiments of the present invention, the 3' end of the gRNA is modified with 2'-O-methylation.

[0026] In some embodiments of the present invention, the gene editing system in step S2 is in the form of a combination or complex of gRNA and nuclease, particularly a complex of gRNA and nuclease, such as CRISPR-Cas9 RNP.

[0027] Specifically, the method of introducing cells in step S2 can be vector transformation, transfection, heat shock, electroporation, transduction, or microinjection; in some embodiments of the present invention, the method of introducing cells is electroporation.

[0028] In some embodiments of the present invention, step S2 includes: mixing nuclease protein and gRNA, incubating, adding the vector described in step S1, mixing with immune cells, and electroporating.

[0029] Specifically, the conditions for electroporation can be 1300V, 20ms, 1Pulse.

[0030] Specifically, immune cells can be T cells (such as NKT cells, γδT cells), NK cells, B cells, macrophages, dendritic cells, monocytes, and especially T cells.

[0031] Specifically, T cells can be CD3 positive.

[0032] Specifically, immune cells can be autologous (e.g., from a subject with a disease requiring gene therapy) or allogeneic (e.g., from a healthy donor).

[0033] Specifically, the immune cells used in step S2 can be obtained through the following steps: PBMC isolation; T cell activation.

[0034] Specifically, the PBMC separation steps may include: diluting the peripheral blood sample, mixing it with lymphocyte separation solution, centrifuging, and aspirating the white blood cell layer (then washing, resuspending, and freezing).

[0035] Specifically, the T cell activation steps may include: activating and culturing PBMCs using CD3 / CD28 beads.

[0036] Specifically, the identification in step S3 may include the identification of gene integration sites, CAR receptor expression identification, and CAR receptor immune activation function identification.

[0037] In a second aspect of the invention, site-specific integrated immune cells prepared in the first aspect are provided, for example, CAR-immune cells (e.g., CAR-T, CAR-NK, CAR-NKT, CAR-γδT cells, especially CAR-T cells) with a gene knocked out at a specific site (e.g., HPK1).

[0038] In some embodiments of the present invention, the cells described above are HPK1 gene knockout CD19 CAR-T cells.

[0039] Specifically, the aforementioned CAR-T cells can be autologous CAR-T cells or universal CAR-T cells.

[0040] In a third aspect of the invention, a CAR gene is provided, comprising a ligand-binding domain, a transmembrane domain, and a co-stimulatory domain.

[0041] Specifically, the ligand-binding domain can target targets selected from the following: ROR1, Her2, Ll-CAM, CD19, CD20, CD22, CEA, hepatitis B surface antigen, folate receptor antibody, CD23, CD24, CD30, CD33, CD38, CD276, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kdr, κ light chain, Lewis Y, L1 cell adhesion molecule (CD171), MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, tumor-fetoprotein antigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, MAGEA3, CE7, Wilms tumor 1 (WT-1), cyclin A1 (CCNA1), BCMA, and interleukin-12 or any combination thereof; in some embodiments of the present invention, the target is CD19, i.e., CD19 CAR.

[0042] Specifically, the transmembrane domain can be selected from the transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0043] Specifically, the co-stimulatory domain can be a co-stimulatory signal transduction domain selected from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1B). B), CD150(SLAMF1), CD152(CTLA4), CD223(LAG3), CD270(HVEM), CD272(BTLA), CD273(PD-L2), CD274(P D-L1), CD276(B7-H3), CD278(ICOS), CD357(GITR), DAP10, LAT, NKG2C, SLP76, PD1, LIGHT, TRIM and ZAP70.

[0044] In one embodiment of the invention, the CAR gene comprises, and in particular, is composed of, the nucleotide sequence shown in SEQ ID NO:17.

[0045] In a fourth aspect of the invention, a vector comprising the CAR gene described in the third aspect is provided.

[0046] Specifically, the vector is a plasmid vector, such as the PUC57 plasmid.

[0047] Specifically, the CAR gene can be linked to the vector via restriction endonuclease (such as EcoRI, BamHI) restriction sites.

[0048] In a fifth aspect of the invention, a nucleic acid molecule targeting HPK1 is provided, comprising, from 5' to 3', a target sequence (TSF), a left homologous arm (LHA), a promoter, a polyA, a right homologous arm (RHA), and the target sequence (TSF).

[0049] Specifically, the TSF contains a nucleotide sequence as shown in SEQ ID NO: 16, or the sequence of the TSF is as shown in SEQ ID NO: 16.

[0050] Specifically, the lengths of the left and right homologous arms are 200-1000 bp (e.g., 200, 300, 400, 500, 600, 700, 800, 900, 1000 bp). In one embodiment of the present invention, the left and right homologous arms respectively contain nucleotide sequences as shown in SEQ ID NO: 9 and 10, or as shown in SEQ ID NO: 9 and 10, respectively. In another embodiment of the present invention, the left and right homologous arms respectively contain nucleotide sequences as shown in SEQ ID NO: 11 and 12, or as shown in SEQ ID NO: 11 and 12, respectively. In another embodiment of the present invention, the left and right homologous arms respectively contain nucleotide sequences as shown in SEQ ID NO: 13 and 14, or as shown in SEQ ID NO: 13 and 14, respectively.

[0051] Specifically, the promoter can be EF1αpromoter, whose nucleotide sequence is shown in SEQ ID NO: 15.

[0052] Specifically, polyA can be BGHpA, SV40 polyA, or WPRE, and their nucleotide sequences are shown in SEQ ID NO: 6-8, respectively; in one embodiment of the present invention, polyA is SV40polyA.

[0053] Specifically, there is also a restriction endonuclease (such as BamHI, EcoRI) cleavage site between the promoter and polyA.

[0054] In a sixth aspect of the invention, a carrier comprising the nucleic acid molecule described in the fifth aspect is provided.

[0055] Specifically, the vector is a plasmid vector, such as the PUC57 plasmid.

[0056] Specifically, the nucleic acid molecules described in the fifth aspect can be linked to the vector via restriction endonuclease (such as SpeI, ApaI) cleavage sites.

[0057] In a seventh aspect of the present invention, a homologous recombination repair template DNA is provided, comprising a target gene and a homologous arm.

[0058] Specifically, the nucleic acid molecule contains, from 5' to 3', the following sequence: target sequence (TSF), left homologous arm (LHA), promoter, target gene, polyA, right homologous arm (RHA), and target sequence (TSF).

[0059] Specifically, the target gene can be a CAR gene, such as that described in the third aspect of this invention.

[0060] Specifically, the TSF contains a nucleotide sequence as shown in SEQ ID NO: 16, or the sequence of the TSF is as shown in SEQ ID NO: 16.

[0061] Specifically, the lengths of the left and right homologous arms are 200-1000 bp (e.g., 200, 300, 400, 500, 600, 700, 800, 900, 1000 bp). In one embodiment of the present invention, the left and right homologous arms respectively contain nucleotide sequences as shown in SEQ ID NO: 9 and 10, or as shown in SEQ ID NO: 9 and 10, respectively. In another embodiment of the present invention, the left and right homologous arms respectively contain nucleotide sequences as shown in SEQ ID NO: 11 and 12, or as shown in SEQ ID NO: 11 and 12, respectively. In another embodiment of the present invention, the left and right homologous arms respectively contain nucleotide sequences as shown in SEQ ID NO: 13 and 14, or as shown in SEQ ID NO: 13 and 14, respectively.

[0062] Specifically, the promoter can be EF1αpromoter, whose nucleotide sequence is shown in SEQ ID NO: 15.

[0063] Specifically, polyA can be BGHpA, SV40 polyA, or WPRE, and their nucleotide sequences are shown in SEQ ID NO: 6-8, respectively; in one embodiment of the present invention, polyA is SV40polyA.

[0064] In an eighth aspect of the invention, a vector comprising the homologous recombination repair template DNA described in the seventh aspect is provided.

[0065] In some embodiments of the present invention, the vector is a vector capable of producing AAV, microcircular DNA, dsDNA or ssDNA, particularly a plasmid vector capable of producing microcircular DNA, such as pMC.BESPX-MCS2.

[0066] In other embodiments of the invention, the vector is AAV, microcircular DNA, dsDNA, or ssDNA, particularly microcircular DNA.

[0067] In a ninth aspect of the present invention, a method for preparing the vector described in the eighth aspect is provided, comprising the steps of sequentially constructing the nucleic acid molecule described in the fifth aspect of the present invention and the target gene described in the third aspect onto the vector.

[0068] Specifically, the method includes the following steps:

[0069] (1) The vector and microcircular parenteral plasmid (such as pMC.BESPX-MCS2) described in the sixth aspect of the present invention are digested with enzymes, ligated, transformed and screened;

[0070] (2) The vector described in the fourth aspect of the present invention and the plasmid obtained by screening in step (1) are digested with enzymes, ligated, transformed and screened.

[0071] Optionally, (3) the plasmid obtained in step (2) is induced to culture with arabinose and the induced plasmid is extracted.

[0072] Specifically, steps (1) and / or (2) also include a step of sequencing identification after ligation.

[0073] Specifically, the steps (1) and / or (2) involve converting the resulting plasmid after ligation into microcircular competent cells (e.g., ZYCY10P3S2TE.coil).

[0074] Specifically, the culture medium for induction culture in step (3) is TB medium, in which the concentration of arabinose is 0.01-1%.

[0075] In a tenth aspect of the invention, a gRNA is provided having a targeting domain complementary to a target sequence at an HPK1 site (e.g., HPK1 EXON).

[0076] In some embodiments of the present invention, the target sequence comprises, or is composed of, any of the nucleotide sequences shown in SEQ ID NO: 1-5.

[0077] In some embodiments of the present invention, the gRNA comprises, and in particular, consists of, the nucleotide sequence shown in SEQ ID NO:18.

[0078] Specifically, the gRNA also includes chemical modifications of the bases, such as methylation or thioation or a combination of both; more specifically, the 5' end and / or the 3' end of the gRNA are modified with 2'-O-methylation and / or 3'-thiosulfate, with each of the three bases containing methylation; in some embodiments of the present invention, the 3' end of the gRNA is modified with 2'-O-methylation.

[0079] In an eleventh aspect of the present invention, a composition is provided comprising the gRNA described in the ninth aspect, and a CRISPR nuclease.

[0080] Specifically, the nuclease can be SpCas9, SaCas9, eSpCas9, Cas12a, Cas13, or cpf1; in some embodiments of the present invention, the nuclease can be SpCas9.

[0081] In a twelfth aspect of the present invention, a complex (RNP) of gRNA and nuclease as described in the ninth aspect is provided.

[0082] Specifically, the nuclease can be SpCas9, SaCas9, eSpCas9, Cas12a, Cas13, or cpf1; in some embodiments of the present invention, the nuclease can be SpCas9.

[0083] In a thirteenth aspect of the present invention, the use of the method described in the first aspect of the present invention and the site-integrated immune cells prepared therefrom, the CAR gene described in the third aspect and the vector containing the CAR gene, the nucleic acid molecule described in the fifth aspect and the vector containing the nucleic acid molecule, the homologous recombination repair template DNA described in the seventh aspect and the vector containing the homologous recombination repair template DNA, the gRNA described in the tenth aspect, the composition described in the eleventh aspect, and the complex described in the twelfth aspect in the preparation of gene therapy drugs are provided.

[0084] Specifically, diseases requiring gene therapy include, for example, malignant tumors, genetic diseases (such as hemophilia, congenital amaurosis, cystic fibrosis), cardiovascular diseases, infectious diseases (such as AIDS), autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, psoriasis), and immune rejection, especially malignant tumors.

[0085] Specifically, malignant tumors can include, for example, lymphoma, chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), acute lymphoblastic leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma (NHL), diffuse large cell lymphoma (DLCL), multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, and medulloblastoma, etc.

[0086] The method provided by this invention includes the design of a homologous recombination repair template targeting a specific site (e.g., HPK1), and the simultaneous introduction of the homologous recombination repair template and a gene editing system into immune cells via physical or chemical means. This achieves gene knockout and target gene introduction in one step, precisely integrating the target gene (e.g., CAR gene) into a specific site (e.g., HPK1) on immune cells, producing cells with specific site gene knockout and stable expression of the target gene. This method is characterized by simple preparation, low cost, stable expression, stable function, and no impact on cell gene characteristics. The resulting cells exhibit high killing efficiency and strong infiltration ability. Attached Figure Description

[0087] Figure 1 The diagram shows a design of a homologous recombination repair template with restriction enzyme sites.

[0088] Figure 2 The image shows the plasmid map of the constructed microcircular vector.

[0089] Figure 3 The results of WB testing show the HPK1 knockout efficiency.

[0090] Figure 4 The results show the positive rate of CD19 CAR detected by flow cytometry.

[0091] Figure 5 The image shows the results of ELISA detection of IFN-γ release levels. Detailed Implementation

[0092] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0093] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.

[0094] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0095] Unless otherwise specified, all techniques or conditions used in the examples were performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained through legitimate channels.

[0096] Example 1: Preparation of microcircular vectors containing target gene CAR

[0097] 1. Using the scFv sequence of the CD19 CAR, the signal peptide, scFv, hinge region, transmembrane region, and co-stimulatory signal region sequences were sequentially assembled, and EcoRI and BamHI restriction sites were designed at both ends of this sequence. The whole genome of the target gene CAR sequence (SEQ ID NO:17) was synthesized according to the above structure (BGI Genomics), and named PUC57-CD19 CAR.

[0098] 2. Design and synthesis of HPK1 site homologous recombination repair template: The method is as follows: An sgRNA sequence is designed on the HPK1 genome EXON, and the HPK1 sgRNA targeting region (SEQ ID NO:16), left homologous arm (SEQ ID NO:9), EF1αpromoter (SEQ ID NO:15), SV40 polyA (SEQ ID NO:7), right homologous arm (SEQ ID NO:10), and sgRNA targeting region (SEQ ID NO:16) are sequentially spliced ​​together. SpeI and ApaiI restriction sites (e.g., ...) are designed at both ends of this sequence. Figure 1 (As shown). The entire genome of the HPK1 site homologous recombination sequence was synthesized according to the above structure (BGI Genomics), and named PUC57-HPK1HA.

[0099] 3. The construction of microcircular vectors containing the target gene CAR is as follows:

[0100] (1) Plasmids were extracted from the above-mentioned PUC57-CD19 CAR, PUC57-HPK1HA and microcircular DNA vector pMC.BESPX-MCS2 provided by the microcircular DNA vector kit using the plasmid extraction kit (TIANGEN).

[0101] (2) PUC57-HPK1 HA and pMC.BESPX-MCS2 were digested with SpeI and ApaI enzymes, detected by gel electrophoresis, and the target vector and fragment were recovered by DNA gel recovery kit (TIANGEN) to obtain the digestion products.

[0102] (3) Ligation reaction: The above-mentioned enzyme-digested and recovered vector and fragment were ligated, transferred to microcircular competent cells (ZYCY10P3S2TE.coil), plated on Kans resistant plates, and cultured overnight.

[0103] (4) Cloning identification: Select single-clone bacteria, extract plasmids using the plasmid miniprep kit (TIANGEN), confirm the correctness by SpeI & ApaI double enzyme digestion electrophoresis, and perform sequencing. The sequencing results show that the gene sequence is correct and the pMC.BESPX-HPK1 HA plasmid was successfully constructed.

[0104] 4. Following the above procedure, construct the target CAR gene into the pMC.BESPX-HPK1 HA vector to construct the pMC.BESPX-HPK1 HA-CD19 CAR plasmid. Sequencing confirmed successful construction. Store the constructed plasmid bacterial culture in glycerol at -80℃ for future use. The plasmid map information is shown below. Figure 2 As shown.

[0105] 5. Extraction of pMC.BESPX-HPK1 HA-CD19 CAR plasmid-induced expression

[0106] (1) The successfully constructed microcircular plasmid glycerol bacteria were inoculated into LB medium containing Kan resistance and incubated at 30°C until OD. 600 ≈0.6.

[0107] (2) Inoculate onto TB medium and incubate overnight with shaking.

[0108] (3) Take a small amount of bacterial solution the next day to measure pH and OD. 600 Maintain pH between 6.5 and 7.5, OD 600 Between 6 and 8. Add TB medium containing 0.1% L-arabinose and induce culture for 4 h. After induction, take a small amount of the induced bacterial culture for small-dose extraction, and detect the quality of pMC.BESPX-HPK1 HA-CD19 CAR plasmid by EcoRI & BamHI restriction enzyme digestion and electrophoresis.

[0109] (4) After the quality is qualified, use QIAGEN EndoFree Plasmid Maxi Kit to extract MC.BESPX-HPK1 HA-CD19 CAR plasmid in large doses according to the operation steps, determine the concentration, and store it in a refrigerator at -20℃ for later use after passing the quality inspection.

[0110] Example 2: Preparation of Cas9 and sgRNA

[0111] 1. The HPK1 sgRNA sequence (SEQ ID NO:18) was designed and obtained from the website https: / / www.genscript.com / grna-design-tool.html, synthesized by Genscript Biosciences, and the three bases at the 3' end of the sgRNA were modified with methylation.

[0112] 2. SpCas9 protein was obtained through prokaryotic expression and purification. First, the prokaryotic expression vector carrying the SpCas9 gene was transformed into the prokaryotic expression strain Transetta (DE3). After IPTG induction, the bacterial cells were collected, broken up, concentrated, and impurities were removed by passing through a cation exchange column. The target protein was then eluted with high salt and dialyzed to 50 mM phosphate buffer. The protein was then concentrated to 2 mg / mL and stored at -80°C for later use.

[0113] Example 3: Preparation of HPK1-based CAR-T cells for targeted integration using microcircular DNA

[0114] 1. PBMC separation

[0115] Peripheral blood samples (PBMCs) were collected from volunteers for separation. The blood sample was diluted with an equal volume of PBS buffer and gently added to Ficoll lymphocyte separation medium. The sample was centrifuged at 800g for 20 min. After centrifugation, the central ring of milky-white lymphocytes was aspirated and transferred to a new 50mL centrifuge tube. After washing twice with PBS, the cells were counted, resuspended in cryopreservation buffer, and aliquoted into 1mL portions for liquid nitrogen cryopreservation.

[0116] 2. T cell resuscitation and activation culture

[0117] Frozen PBMCs from healthy volunteers were revived in a 37°C water bath, washed twice with 1640 medium, and counted. Cells were resuspended in 100 U / mL IL-2 + 10% FBS + X-VIVO-15. CD3 / CD28 beads were added for activation culture for 72 h.

[0118] 3. Preparation of HPK1 cells by electroporation of microcircular DNA into T cells KO CD19 CART cells

[0119] (1) Cell treatment: T cells activated for 72 hours were collected into 15 mL centrifuge tubes, washed once with PBS, and then magnetic beads were removed from a magnetic rack. Cells were counted and the cell density was adjusted to 1.0 × 10⁶ cells using electroporation buffer. 7 The cells / mL were used for electroporation.

[0120] (2) Electroporation system preparation: After mixing the Cas9 protein and gRNA (prepared in Example 2) evenly, incubate at room temperature for 10 min, add the microcircular plasmid to be electroporated, mix with 100 μL of cell suspension, and perform electroporation.

[0121] (3) Electroporation: The Invitrogen Neon (Thermo Fisher) electroporation system was used for electroporation at 1300V, 20ms, 1Pulse. The cell suspension was then transferred to preheated 300U / mL IL-2 + 10% FBS + X-VIVO-15 medium and incubated at 37℃ in a 5% CO2 incubator.

[0122] 4. Preparation of HPK1 by electroporation of T cells with Cas9 / sgRNA ribonucleoprotein (Cas9-RNP, hereinafter referred to as RNP) KO T cells

[0123] (1) Cell processing: T cells activated for 72 hours were collected in 15 mL centrifuge tubes, washed once with PBS, and then magnetic beads were removed from a magnetic rack and counted. The cell density was adjusted to 1.0 × 10⁶ cells using electroporation buffer. 7 The cells / mL were used for electroporation.

[0124] (2) Electroporation system preparation: After mixing the Cas9 protein and gRNA (prepared in Example 2) evenly, incubate at room temperature for 10 min, mix with 100 μL of cell suspension, and then perform electroporation.

[0125] (3) Electroporation: The Invitrogen Neon (Thermo Fisher) electroporation system was used for electroporation at 1300V, 20ms, 1Pulse. The cell suspension was then transferred to preheated 300U / mL IL-2 + 10% FBS + X-VIVO-15 medium and incubated at 37℃ in a 5% CO2 incubator.

[0126] 5. Preparation of HPK1 cells by lentiviral infection combined with RNP electroporation of T cells KO CD19 CART cells (Lenti)

[0127] (1) Lentiviral infection of T cells: T cells cultured for 48 hours were activated and infected with lentivirus. Electroporation was performed 24 hours after infection.

[0128] (2) Electroporation treatment: CAR-T cells infected for 24 hours were collected into 15 mL centrifuge tubes, washed once with PBS, and then magnetic beads were removed from a magnetic rack. Cells were counted. The cell density was adjusted to 1.0 × 10⁶ cells / mL using electroporation buffer. 7 The cells / mL were used for electroporation.

[0129] (3) Electroporation system preparation: After mixing Cas9 protein and gRNA (prepared in Example 2) evenly, incubate at room temperature for 10 min, mix with 100 μL of cell suspension, and perform electroporation.

[0130] (4) Electroporation: The Invitrogen Neon (Thermo Fisher) electroporation system was used to perform electroporation at 1300V, 20ms, 1Pulse. The cell suspension was then transferred to preheated 300U / mL IL-2 + 10% FBS + X-VIVO-15 medium and incubated at 37℃ in a 5% CO2 incubator.

[0131] 6. Western blotting was used to detect the HPK1 knockout efficiency of CD19-targeted CAR-T cells.

[0132] (1) Total protein extraction and concentration detection: T cells, HPK1 knockout T cells (HPK1KO T cells), and CAR-T cells (HPK1 KO CD19 CART cells) were collected and washed twice with 1 mL PBS. 20 μL RIPA was added to each sample, and the cells were lysed at 4℃ for 1 h. Centrifuged at 12000g for 15 min at 4℃, the supernatant was collected, and the total protein concentration was detected by the BCA method.

[0133] (2) Electrophoresis: 20 μg of protein was loaded onto the sample and run for SDS-PAGE electrophoresis.

[0134] (3) Transfer: 300mA, 40min to transfer to PVDF membrane.

[0135] (4) Sealing: Sealing with 5% skim milk for 2 hours.

[0136] (5) Incubation of primary antibodies: Prepare antibodies, HPK1 primary antibody and GAPDH primary antibody diluted 1:1000 and incubated at 4°C overnight.

[0137] (6) Washing the membrane: Discard the primary antibody incubation solution and wash the membrane three times with TBST solution for 5 minutes each time.

[0138] (7) Secondary antibody incubation: Dilute the secondary antibody 1:5000 and incubate at room temperature in the dark for 45 min.

[0139] (8) Washing the membrane: Wash the membrane 4 times with TBST, 10 minutes each time.

[0140] (9) Color development: ECL luminescence reagent kit for color development, photography, and data processing.

[0141] The results are as follows Figure 3 As shown, the HPK1 knockout efficiency of cells by electroporation of microcircular plasmids and RNPs simultaneously is about 80%, which is not significantly different from that of electroporation of RNPs alone.

[0142] 7. Flow cytometry detection efficiency of targeting the HPK1 gene site for CD19 CAR gene knock-in

[0143] Each group was sampled at 2.0 × 10⁻⁶. 6 Cells were washed twice with 1 mL of 2% BSA / PBS and aliquoted into 1.5 mL EP tubes (50 μL each), designated as the NC, secondary antibody-stained control, and experimental groups, respectively. 5 μL of Biotinylated-CD19 protein was added to the experimental group tubes, and the cells were incubated at 4°C in the dark for 1 h, followed by washing twice with 1 mL of 2% BSA / PBS. Cells were resuspended in 50 μL of 2% BSA / PBS. PE-Streptavidin secondary antibody was added to both the control and experimental groups, and the cells were incubated at 4°C in the dark for 30 min. After incubation, cells were washed twice with 1 mL of 2% BSA / PBS, and then resuspended in 200 μL of 2% BSA / PBS. The percentage of CD19+ cells was then analyzed.

[0144] The results are as follows Figure 4 As shown, the HPK1 knock-in efficiency of the CAR gene is about 30%, which is not much different from the transduction efficiency of ordinary lentiviruses.

[0145] 8. In vitro killing experiment of the prepared CD19-targeted CAR-T cells on tumor cells.

[0146] First, resting T cells, HPK1 knockout T cells (HPK1 KO T cells), CAR-T cells (HPK1 KO CD19 CART cells), and target cells K562, Daudi, and Raji were collected in 15 ml centrifuge tubes. The cells were washed twice with additive-free X-VIVO-15 medium, the supernatant was discarded, and the cells were resuspended in additive-free X-VIVO-15 medium and adjusted to a specific density. Effector cells and target cells were mixed at a 1:1 ratio, with each sample prepared in triplicate. The mixture was incubated in additive-free X-VIVO-15 medium at 37°C for 16 h, followed by centrifugation at 300 g for 5 min. The supernatant was collected for IFN-γ release level detection (IFN-γ ELISA kit: purchased from Beijing Yiqiao Shenzhou Co., Ltd., catalog number: KIT11725A).

[0147] The results are as follows Figure 5 As shown, the IFN-γ release level of the HPK1 KO CD19 CART cell group was higher than that of the T cell and HPK1 KO T cell groups, showing a significant killing trend. The difference was not significant compared with the lentivirus infection group (HPK1 KO CD19 CART cells (Lenti)), further proving that CD19CAR knock-in was successful.

[0148] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0149] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0150] Listing the steps of the method in a certain order in this invention does not constitute any restriction on the order of the method steps. sequence list <110> Xi'an Yufan Biotechnology Co., Ltd. <120> A method for targeted integration of a target gene into a specific site on immune cells and its application. <130> 1 <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 gacctggtgg cactgaaga 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 gctcgagaca aggtgtcag 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <400> 3 aaggtgtcag gggacctgg 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 accactatga cctgctacag 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gacctgctac agcggctggg 20 <210> 6 <211> 66 <212> DNA <213> Artificial Sequence <400> 6 ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60 ggacct 66 <210> 7 <211> 122 <212> DNA <213> Artificial Sequence <400> 7 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120 ta 122 <210> 8 <211> 591 <212> DNA <213> Artificial Sequence <400> 8 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc 420 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc t 591 <210> 9 <211> 800 <212> DNA <213> Artificial Sequence <400> 9 cctgcgcagt gcagaggagg taacccaagt gcatgtatgt gacaggtgaa caggcatgta 60 cactgcatag ccatcccttt gcagacctgc cgccttcggg ttgtggccaa accttcaagc 120 acccagcaga ggtttagaca ctacctagga gacgggggcg ggggttctaa ggcacccccc 180 tccaacccag ttacgattaa gatcatttgc atcacattta catccagccc tgggccttgg 240 aggttgaggg tggtcccttg ggtcccaaga caagctgccc ctcagaggct ctcagagccc 300 aggggacggg ggggtggagc ccgttgcaca gtcgtgcagt gcagctgggg caggaagcga 360 gagtgaggag gggggaggcc acagcccgcg gaggcaaggc gggtgcaggg cttctgggga 420 cggagggagg tgccagaagt tgagccctga ggccctgctg gcccctgggc gcaggcccag 480 ctcaggcccc cagggatgga cgtcgtggac cctgacattt tcaatagaga cccccgggac 540 cactatgacc tgctacagcg gctgggtggc ggcacgtatg gggaagtctt taaggtgagg 600 aagcccctct gtccccaaca ccctacaacc agtgaccttt aaggcaagcc ctcggcaggg 660 aggaggctgc aagagggggt cccagttctt gaccttgact cagggatgct gtggagggac 720 tctggcaaat ggagcctgcc tgactgccaa cttccccttt gcaaggctcg agacaaggtg 780 tcaggggacc tggtggcact 800 <210> 10 <211> 800 <212> DNA <213> Artificial Sequence <400> 10 gaagatggtg aagatggagc ctggtgagga gggaacatgg agctccttcc ccacatcccc 60 accttcgcct cctgggacac tgggatcacc tcccctgtag ctctctcccc atgcgagctg 120 ccttttctct cccgccctcc tcacagatga tgatgtctcc acccttcaga aggaaatcct 180 catattgaaa acttgccggc acgccaacat cgtggcctac catgggagtt atctctggtg 240 atgaacctca gacccaccct ggcacccagc ccagccccta acccggagtc cccatttctt 300 gatgggggta ccctcagttc tttttttttc ttttcttttt cttttttgag atggagtctt 360 gttctgttgc caggctggag tgcagtggca tgatctcagc tcactgcaac ctctgcctcc 420 tgggttcaag caattctctg cctcagactc ccgagtagct gggattacag gcgctcacca 480 ccctgcccgg caaatttttg tatttttagt agagatgggg tttcaccatc ttggccaggc 540 tggtcttgaa ctcctgacct tgtgatccac tcgcctcggc ctccgaaagt gctgggatta 600 caggtgtgat ccaccgcgcc tggtcttggc accctcagtt ctaaccttga acttccattt 660 ctcaccataa cccgacctcc catcctggac ccccattttt gactgggcta accctaccat 720 gatcctcact cctaagtccc acttttctga ccagagcagc ctctaatcta atctccaatc 780 taaaattccc atttctgacc 800 <210> 11 <211> 400 <212> DNA <213> Artificial Sequence <400> 11 gggtgcaggg cttctgggga cggagggagg tgccagaagt tgagccctga ggccctgctg 60 gcccctgggc gcaggcccag ctcaggcccc cagggatgga cgtcgtggac cctgacattt 120 tcaatagaga cccccgggac cactatgacc tgctacagcg gctgggtggc ggcacgtatg 180 gggaagtctt taaggtgagg aagcccctct gtccccaaca ccctacaacc agtgaccttt 240 aaggcaagcc ctcggcaggg aggaggctgc aagagggggt cccagttctt gaccttgact 300 cagggatgct gtggagggac tctggcaaat ggagcctgcc tgactgccaa cttccccttt 360 gcaaggctcg agacaaggtg tcaggggacc tggtggcact 400 <210> 12 <211> 400 <212> DNA <213> Artificial Sequence <400> 12 gaagatggtg aagatggagc ctggtgagga gggaacatgg agctccttcc ccacatcccc 60 accttcgcct cctgggacac tgggatcacc tcccctgtag ctctctcccc atgcgagctg 120 ccttttctct cccgccctcc tcacagatga tgatgtctcc acccttcaga aggaaatcct 180 catattgaaa acttgccggc acgccaacat cgtggcctac catgggagtt atctctggtg 240 atgaacctca gacccaccct ggcacccagc ccagccccta acccggagtc cccatttctt 300 gatgggggta ccctcagttc tttttttttc ttttcttttt cttttttgag atggagtctt 360 gttctgttgc caggctggag tgcagtggca tgatctcagc 400 <210> 13 <211> 200 <212> DNA <213> Artificial Sequence <400> 13 aagcccctct gtccccaaca ccctacaacc agtgaccttt aaggcaagcc ctcggcaggg 60 aggaggctgc aagagggggt cccagttctt gaccttgact cagggatgct gtggagggac 120 tctggcaaat ggagcctgcc tgactgccaa cttccccttt gcaaggctcg agacaaggtg 180 tcaggggacc tggtggcact 200 <210> 14 <211> 200 <212> DNA <213> Artificial Sequence <400> 14 gaagatggtg aagatggagc ctggtgagga gggaacatgg agctccttcc ccacatcccc 60 accttcgcct cctgggacac tgggatcacc tcccctgtag ctctctcccc atgcgagctg 120 ccttttctct cccgccctcc tcacagatga tgatgtctcc acccttcaga aggaaatcct 180 catattgaaa acttgccggc 200 <210> 15 <211> 212 <212> DNA <213> Artificial Sequence <400> 15 gggcagagcg cacatcgccc acagtcccga agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gccttttttcc cgagggtggg ggagaaccgt fathergtgc agtagtcgcc gtgaacgttc 180 tttttcgcaa cggggtttgcc gccagaacac ag <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <400> 16 ggacctggtg gcactgaaga tgg <210> 17 <211> 1464 <212> DNA <213> Artificial Sequence <400> 17 gccaccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc gccaggccgg acatccagat gacacagact acatcctccc tgtctgcctc tctgggagac 120 180. 180. 180. 180. 180. 180. 180. 180. 180 cagaaaccag atggaactgt taaactcctg atctaccata catcaagatt acactcagga 240 gtcccatcaa ggttcagtgg cagtgggtct ggaacagatt attctctcac cattagcaac 300 ctggagcaag aagatattgc cacttacttt tgccaacagg gtaatacgct tccgtacacg 360 ttcggagggg ggaccaagct ggagatcaca ggtggcggtg gctcgggcgg tggtgggtcg 420 ggtggcggcg gatctgaggt gaaactgcag gagtcaggac ctggcctggt ggcgccctca 480 cagagcctgt ccgtcacatg cactgtctca ggggtctcat tacccgacta tggtgtaagc 540 tggattcgcc agcctccacg aaagggtctg gagtggctgg gagtaatatg gggtagtgaa 600 accacatact ataattcagc tctcaaatcc agactgacca tcatcaagga caactccaag 660 agccaagttt tcttaaaaat gaacagtctg caaactgatg acacagccat ttactactgt 720 gccaaacatt attactacgg tggtagctat gctatggact actggggtca aggaacctca 780 gtcaccgtct cctcaaccac gacgccagcg ccgcgcccac caacaccggc gcccaccatc 840 gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgccgtg 900 cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgcccct ggccgggacc 960 tgtggggtcc tgctcctgag cctggtgatc accctgtact gcaagcgggg ccgcaagaag 1020 ctcctgtaca tcttcaagca gccatttatg cgcccagtgc agaccaccca ggaggaggat 1080 ggctgctcgt gccgcttccc agaggaggaa gagggcggct gtgagctgcg cgtgaagttc 1140 agcaggagcg ccgacgcccc cgcgtacaag cagggccaga accagctcta caacgagctc 1200 aacctgggcc gccgcgagga gtacgatgtg ctggacaagc gccgcggccg ggaccctgag 1260 atggggggca agccgcgcag gaagaaccct caggagggcc tgtacaacga gctgcagaag 1320 gataagatgg cggaggccta cagcgagatc gggatgaagg gcgagcgccg gaggggcaag 1380 gggcacgatg gcctgtacca gggcctcagc acagccacca aggacaccta cgacgccctg 1440 cacatgcagg ccctgccccc tcgc 1464 <210> 18 <211> 99 <212> DNA <213> Artificial Sequence <400> 18 ggacctggtg gcactgaaga gttttagagc tagaaatagc aagttaaaat aaggctagtc 60 cgttatcaac ttgaaaaagt ggcaccgagt cggtgcttt 99

Claims

1. A method for targeted integration of a target gene into a specific site on immune cells, wherein the method achieves gene knockout at the specific site and introduction of the target gene in one step, comprising the following steps: S1, construct a homologous recombination repair template vector targeting a specific site, wherein the homologous recombination repair template comprises, from 5' to 3', the following in sequence: target sequence, left homologous arm, promoter, target gene, polyA, right homologous arm, and target sequence; S2, the gene editing system and the homologous recombination repair template vector obtained in step S1 are introduced into immune cells; in, The specific site is HPK1; the homologous recombination repair template vector is microcircular DNA; the target sequence contains the nucleotide sequence shown in SEQ ID NO: 16; the left and right homologous arms contain the nucleotide sequences shown in SEQ ID NO: 9 and 10, respectively, or as shown in SEQ ID NO: 9 and 10, respectively; The gene editing system is a CRISPR system, which includes gRNA and nuclease; The nuclease used in the CRISPR system is SpCas9; The gRNA comprises, or is composed of, the nucleotide sequence shown in SEQ ID NO: 18; The target gene is the CAR gene; The immune cells mentioned are T cells.

2. The method as described in claim 1, characterized in that, The CAR gene contains a nucleotide sequence as shown in SEQ ID NO:

17.

3. The method as described in claim 1, characterized in that, The promoter is the EF1α promoter.

4. The method as described in claim 1, characterized in that, The polyA is BGHpA, SV40 polyA, or WPRE.

5. The method as described in claim 1, characterized in that, The gRNA also includes chemical modifications of the bases.

6. The method as described in claim 5, characterized in that, The chemical modification is methylation, thiolation, or a combination of both.

7. The method as described in claim 1, characterized in that, The gRNA is modified with 2'-O-methylation and / or 3'-thiosulfate for 1-5 bases at the 5' and / or 3' ends.

8. The method as described in claim 1, characterized in that, The gene editing system described in step S2 is a complex of gRNA and nuclease.

9. The method as described in claim 8, characterized in that, The complex is RNP.

10. The method as described in claim 1, characterized in that, The method of introducing immune cells in step S2 is selected from: vector transformation, transfection, heat shock, electroporation, transduction, and microinjection.

11. The method as described in claim 1, characterized in that, The method further includes step S3, which involves culturing and identifying the immune cells obtained in step S2.

12. The method according to any one of claims 1-11, characterized in that, The immune cells are of autologous or allogeneic origin.

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