CN107746831A

CN107746831A - Universal CART/TCRT cells and its construction method with chemotherapeutic drug resistance

Info

Publication number: CN107746831A
Application number: CN201711082910.9A
Authority: CN
Inventors: 贺小宏; 王延宾; 袁鹏
Original assignee: Nanjing North Heng Biological Technology Co Ltd
Current assignee: Nanjing North Heng Biological Technology Co Ltd
Priority date: 2017-11-07
Filing date: 2017-11-07
Publication date: 2018-03-02
Anticipated expiration: 2037-11-07
Also published as: CN107746831B

Abstract

The invention belongs to genetic engineering and synthetic biology field, more particularly to a kind of universal CART/TCRT cells and its construction method with chemotherapeutic drug resistance, the universal CART/TCRT cells are the allogeneic T cells for having Chimeric antigen receptor and φt cell receptor, and φt cell receptor α, β chain and deoxycytidine kinase enzyme molecule are knocked；The knockout technology of φt cell receptor α, β chain and deoxycytidine kinase enzyme molecule is CRISPR technologies；The universal CART/TCRT cells had both remained the targeting of tumour specific antigen, the problem of eliminating GvHD again and repelling, and weaken the susceptibility to chemotherapeutics Fludarabine and Clofarabine, can be as a kind of universal simple, low cost, high activity CART/TCRT cell preparations use.

Description

Universal CART/TCRT cells and its construction method with chemotherapeutic drug resistance

Technical field

The invention belongs to genetic engineering and synthetic biology field, and in particular to a kind of general with chemotherapeutic drug resistance Type CART/TCRT cells and its construction method.

Background technology

Chimeric antigen receptor T cell (CART) is one of tumour immunotherapy most promising at present, and it is substantially former Reason mainly extract patient itself T cell, and by gene and cell engineering means make its expression specificity chimeric antigen by Body, enable to identify and with reference to tumor cell surface antigen, so as to play a part of target killing tumor cell.CAR-T exists The effect of notable, on August 30th, 2017, the food sanitation safe committee of U.S. FDA a surnames are obtained in leukaemia and lymphoma treating Cloth ratifies Novartis CAR-T cell therapy official listings, for treating recurrent or the leaching of intractable children and adolescents B- cell acutes Bar chronic myeloid leukemia (rALL), its trade name Kymriah (tisagenlecleucel).ALL is 15 25% is accounted in year following childhood cancer confirmed cases, is the most common children with cancer in the U.S., acute lymphocytic is white in China Blood disease accounts for the 80% of acute leukemia, and the incidence of disease is ten a ten thousandths.Also adult can be betided, it is white to account for all adults The 20% of the blood disease incidence of disease.

Estimated according to National Cancer Institute, there are about patient of 3100 ages below 20 years old every year and be diagnosed as This disease, due to the population base that China is huge, there are about 10000 children and youth to suffer from acute leukemia every year.Effectively Therapeutic choice it is extremely limited, in multiple relapse or intractable B- cell Acute Lymphoblastic Leukemias children and adolescent patient In, disease-free survival rate is less than 10%-30% within 5 years.Kymriah security and validity obtains in multi-center clinical trial Confirm, it is 83% to treat the overall relief rate in three months, it is shown that the effect of its is remarkable.

Except B cell ALL, CAR-T is in relapsed or stubborn chronic lymphocytic leukemia (rCLL), also achieved in the treatment of recurrent or Refractory Multiple Myeloma (rNHL) and NHL (NHL) The effect of notable.Diffusivity large B cell lymphoid tumor (DLBCL), conversion follicular lymphoma (TFL), Yi Jiyuan are treated at one Hair property mediastinal B-cell lymphoma (PMBCL) these three NHLs, in entitled ZUMA-1 2 clinical trial phases, Kite CAR-T therapies reached Major Clinical terminal on objective remission rate (objective response rate).Receiving list After secondary axicabtagene ciloleucel infusion, there is up to 82% patient to occur alleviating.It it is 8.7 in median In the follow-up of the moon, the patient for having 44% is still in the paracmasis, and the patient for having 39% is in complete incidence graph.In Bluebird The key name that Bio companies are carried out is 100% patient in two dosage groups in the experiment of b2121 treatment Huppert's disease (n=6) objective reaction is realized；Two patients are negative in MRD；General reaction rate (ORR) is 78%.6 and during follow-up in 4 months, Two patients reach the complete reaction (CR) of stricti jurise.

In addition to CART, φt cell receptor (TCR) T cell therapy also achieves considerable progress.TCRT cell technologies are earliest From tumor-infiltrated T cell (TIL), this kind of T cell " would generally invade " cancerous tissue, and this illustrates that they have necessarily to cancer cell Recognition capability.Fact-finding TIL has special to cancer cell related antigen (Tumor Associated Antigen, TAA) Property recognition capability.This kind of antigen includes CEA, Her-2, CD19, gp100, MART-1, MAGA-A3, NY-ESO-1, etc..These TAA has the expression of relative particularity in different cancers, thus also turns into the object of attack of immune system.Nevertheless, These natural TIL recognition capability is generally weaker, therefore can not form the favourable attack to cancer cell.In this case, may be used Modification is oriented to reach the purpose of attack tumour to import TIL specific TCR by external source to T cell.2016《From So-medical science》The exciting TCR treatment cases of magazine ran numerical example, this clinical test cured by Univ Maryland-Coll Park USA A entitled " the TCR " of gene modification of institute, medical college of University of Pennsylvania and immunization therapy company Adaptimmune cooperative research and development Immunotherapeutic.After it have modified several key amino acids, these genes modification TCR substantially increase it is normal with one kind The cancer TAA, NY-ESO-1 that see affinity, and then the cancer to NY-ESO-1 overexpressions is significantly improved, such as it is multiple The therapeutic effect of property myeloma (Multiple Myeloma).In current clinical test, 80% multiple myeloma patients Good clinical response is generated, wherein 70% patient reaches completely or nearly complete response, average progression free survival phase reaches By 19 months.The treatment for other cancers of NY-ESO-1 overexpressions is being carried out successively at present.

But because CART/TCRT is a kind of personalized method of height, everyone cell and the treatment received Difference, many patients receive CART/TCRT treatment before received various treatment methods, as bone marrow transplant, chemotherapy, Targeted therapy etc., the preparation and programming of the T cell obtained also can be different.This individualized treatment mode consumes substantial amounts of people Power material resources, so price is high.The CART cell therapy products that the Kymriah of Novartis ratifies as first FDA, it is priced at 47.5 ten thousand U.S. dollars, the medical expense of such great number, some patient families can be caused to be difficult to bear, while also greatly limit its city Field application.

The CART/TCRT first echelons including Novartis, Kite pharmacy, Juno are using autotransplantation side at present Method prepares CART/TCRT cells, and carrying out treatment using allogeneic T cells preparation CART/TCRT cells provides another thinking, but The row for being due to donor T-cells to the graft-versus-host reaction (GvHD) of host and host immune system to allogeneic T cells Reprimand is removed, and significantly limit its application.

The TCR of CART cell surfaces is knocked out by way of gene editing can eliminate because allogeneic T cells are to host's Identification and caused by GvHD, be used to prepare universal CART cells to a certain extent.Currently used gene editing instrument Mainly there are TALEN and CRISPR.

TAL effectors (TAL effector, TALE) are most early in phytopathogen Xanthomonas campestris (Xanthomonas Sp.) it is found in the invasion and attack infection research to plant.Because TALE has the specific binding capacity of DNA sequence dna, follow-up study FokI DNA nucleases and artificial T ALE are connected by engineering science means, formd a kind of with specific gene group volume Collect the strong tools of function, i.e. TALEN.

Typical TALEN by nuclear localization sequence (Nuclear localization signal, NLS) N-terminal domain, The central domain of the series connection TALE repetitive sequences of recognizable specific dna sequence, there is the C-terminal knot of FokI endonuclease functions Structure domain forms.In recent years, TALEN is widely used to yeast, animal and plant cells, and arabidopsis, drosophila, zebra fish and mouse Etc. the research of all kinds of model organisms.

Substantial amounts of molecular cloning and sequencing procedures are needed due to single TALEN modules are carried out into assembling, it is very cumbersome, and And because TALEN structure needs to carry out cumbersome and complicated protein engineering design and optimization according to target dna sequence, so It just substantially prolongs the experimental period of structure TALEN elements.Simultaneously and because TALEN knockout efficiency is influenceed by DNA sequence dna It is larger, and by these many protein deliveries into the cell for multiple genetic operation simultaneously right and wrong often with challenging, Therefore it is very big to limit its application.

Except above-mentioned technological difficulties, because shearings of the TALEN to DNA needs two FokI cutting domains to form dimerization Body, and need at least one identification domain to combine targeting DNA.Although DNA identifies domain with stronger sequence specific Property, but because TALEN shear history not fully relies on the formation of homodimer, so once forming heterodimeric Body, just it is likely to cause effect of missing the target, and DNA mispairing and sequence may finally be caused to change, produces stronger cytotoxicity. When these harmful effects accumulation is excessive, during the scope born more than cellular repair mechanisms, the apoptosis of cell will be caused.It is all above More limitations cause TALEN to be difficult to popularization and application.

CRISPR/Cas systems as a kind of newest genome edit tool, know by the specific DNA that can complete RNA guiding Not and edit.CRISPR/Cas technologies use the guide RNA molecule (sequence-specific of one section of sequence-specific Guide RNA) guiding endonuclease is at target spot, so as to complete the editor of genome.The exploitation of CRISPR/Cas systems is Build more efficient gene site-directed modification technique and provide brand-new platform.

CRISPR/Cas technologies, which have been broken away from, synthesizes and assembles the cumbersome behaviour with specific DNA recognition capability module Make, its gRNA design and synthetic work amount are far smaller than the building process of the DNA identification modules of TALEN and ZFN technologies, and poison Property is well below ZFN technologies.2013《Science》Magazine has delivered two respectively from the significant of MIT and Harvard CRISPR technical papers, independently completed first using CRISPR technologies to human genome editor.Due to CRISPR The easy and efficient characteristic of system, its application have obtained great popularization.Except gene knockout, CRISPR systems are engineered More extensive field is applied to, including gene suppresses, gene activation, gene simple point modification, epigenetic modification, light science of heredity Field, the CRISPR very big research range that must have expanded biology of use.

Huge applications prospect and possible commercial interest due to CRISPR gene editing technologies, foreign countries are with the technology at present Based on obtain huge risk investment biotech firm mainly have Editas Medicine, CRISPR Therapeutics, Intellia Therapeutics etc., master plan carry out related human genetic disease such as retina degenerative disease, genetic muscle The pilot study of the prevention and control field such as disease and hematologic disease.

CAR-T cells (Chimeric antigen receptor-T) are current as immune cell therapy treatment tumour One of tumour immunotherapy hot research field.PD1 (Programmed death 1) be used as a kind of immunosuppression molecule, into For effective target molecule of important oncotherapy, 2016 using CRISPR technologies studies have reported that knock out the PD1 on CART cells Molecule, therapeutic effect of the CART cells to solid tumor can be significantly improved.Research report in 2017, using CRISPR technologies, into Work(builds the CAR-T cells of the gene site-directed insertion CAR genes of mouse TRAC, can significantly extend the modification mouse survival time.

Cellectis companies knock out the intracellular TCRa and CD52 molecules of CART using TALEN technologies, have been applied to face Bed research, but because TALEN that they the use T cell edited all generates serious chromosomal shift phenomenon, this is to it Clinical practice is caused greatly into knurl risk, but the high degree of specificity due to CRISPR in T cell, is compiled using CRISPR The universal CART cells collected carry out oncotherapy, have very big advantage.Simultaneously because TALEN is to the poorly efficient of CART cells Rate editor, purifying can be carried out to the CART cells of gene editing and causes difficulty, while can be because the separation of TCR positive cells is not clean And increase GvHD risk.Our invention causes CRISPR efficiently to edit CART cells, largely favorably In the production of universal CART cells.

Individually knockout TCR α, β chains can not generate universal CART cells, because this CART cells still have HLA molecules are in cell surface expression, so as to be identified and repelled by recipient immune system T cell, it is difficult to reach the purpose for the treatment of.Together When edit TCR α, β chains and suppress molecule, such as PD1, TIM3, CTLA4 can not produce universal CART cells.

The content of the invention

The present invention solves above-mentioned technical problem present in prior art, there is provided a kind of general with chemotherapeutic drug resistance Type CART/TCRT cells and its construction method.

To solve the above problems, technical scheme is as follows：

Universal CART/TCRT cells with chemotherapeutic drug resistance, the universal CART/TCRT cells be have it is embedding The allogeneic T cells of antigen receptor and φt cell receptor are closed, φt cell receptor α, β chain and deoxycytidine kinase enzyme molecule are knocked.

Preferably, the Chimeric antigen receptor is targeting CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, AFP, Alpha folate receptor, CEA, PSCA, PSMA, Her2, EGFR, IL13R α 2, GD2, NKG2D, EGFRvIII, CS1, BCMA, Mesothelin Chimeric antigen receptor.

Preferably, the φt cell receptor is targeting HBV, HPV E6, NY-ESO, mNY-ESO, WT1, MART-1, MAGE- A3, MAGE-A4, P53, Thyroglobulin, Tyrosinase φt cell receptor.

The construction method of universal CART/TCRT cells with chemotherapeutic drug resistance, using CRISPR technologies in allosome T φt cell receptor α, β chain and deoxycytidine kinase (dCK) molecule on intracellular specific knockdown T cell surface.

Preferably, it is as follows using sgRNA and crRNA sequences used in CRISPR technologies：

SgRNA-TCR α are selected from following sequence：

sgRNA-TCRα-1:GAGAATCAAAATCGGTGAAT(SEQ ID NO.1)

sgRNA-TCRα-2:GCTGGTACACGGCAGGGTCA(SEQ ID NO.2)

sgRNA-TCRα-3:GTCAGGGTTCTGGATATCTG(SEQ ID NO.3)

sgRNA-TCRα-4:ACAAAACTGTGCTAGACATG(SEQ ID NO.4)

sgRNA-TCRα-5:CTTCAAGAGCAACAGTGCTG(SEQ ID NO.5)

sgRNA-TCRα-6:TGGAATAATGCTGTTGTTGA(SEQ ID NO.6)

sgRNA-TCRα-7:TAGGCAGACAGACTTGTCAC(SEQ ID NO.7)

sgRNA-TCRα-8:TGGATTTAGAGTCTCTCAGC(SEQ ID NO.8)

sgRNA-TCRα-9:TCTCTCAGCTGGTACACGGC(SEQ ID NO.9)

sgRNA-TCRα-10:AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10)

SgRNA-TCR β are selected from following sequence：

sgRNA-TCRβ-1:GAGCAGCCGCCTGAGGGTCT(SEQ ID NO.11)

sgRNA-TCRβ-2:GAGCTGGTGGGTGAATGGGA(SEQ ID NO.12)

sgRNA-TCRβ-3:GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13)

sgRNA-TCRβ-4:GGCTCTCGGAGAATGACGAG(SEQ ID NO.14)

sgRNA-TCRβ-5:GGAGAATGACGAGTGGACCC(SEQ ID NO.15)

sgRNA-TCRβ-6:GGCGCTGACGATCTGGGTGA(SEQ ID NO.16)

sgRNA-TCRβ-7:GTTGCGGGGGTTCTGCCAGA(SEQ ID NO.17)

sgRNA-TCRβ-8:GCAGTATCTGGAGTCATTGA(SEQ ID NO.18)

sgRNA-TCRβ-9:GGCACACCAGTGTGGCCTTT(SEQ ID NO.19)

sgRNA-TCRβ-10:GGCTCAAACACAGCGACCTC(SEQ ID NO.20)

SgRNA-dCK is selected from following sequence：

sgRNA-dCK-1:GAACATCGGTAAGGAGCCTC(SEQ ID NO.21)

sgRNA-dCK-2:TTAAACAATTGTGTGAAGAT(SEQ ID NO.22)

sgRNA-dCK-3:ATTGTGTGAAGATTGGGAAG(SEQ ID NO.23)

sgRNA-dCK-4:ATCAAGAAAATCTCCATCGA(SEQ ID NO.24)

sgRNA-dCK-5:GTCTTTCTCAGCCAGCTCTG(SEQ ID NO.25)

sgRNA-dCK-6:TTCCTGAACCTGTTGCCAGA(SEQ ID NO.26)

sgRNA-dCK-7:GTAAGGAGCCTCCGGAAATG(SEQ ID NO.27)

CrRNA-TCR α are selected from following sequence：

crRNA-TCRα-1:GAGTCTCTCAGCTGGTACACGGC(SEQ ID NO.28)

crRNA-TCRα-2:CATGTGCAAACGCCTTCAACAAC(SEQ ID NO.29)

crRNA-TCRα-3:CACATGCAAAGTCAGATTTGTTG(SEQ ID NO.30)

crRNA-TCRα-4:TTGCTCCAGGCCACAGCACTGTT(SEQ ID NO.31)

crRNA-TCRα-5:TCTGTGATATACACATCAGAATC(SEQ ID NO.32)

crRNA-TCRα-6:TGACACATTTGTTTGAGAATCAA(SEQ ID NO.33)

crRNA-TCRα-7:TTTGAGAATCAAAATCGGTGAAT(SEQ ID NO.34)

crRNA-TCRα-8:AGAATCAAAATCGGTGAATAGGC(SEQ ID NO.35)

crRNA-TCRα-9:ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.36)

CrRNA-TCR β are selected from following sequence：

crRNA-TCRβ-1:GGTGTGGGAGATCTCTGCTTCTG(SEQ ID NO.37)

crRNA-TCRβ-2:CCTCGGTGTCCTACCAGCAAGGG(SEQ ID NO.38)

crRNA-TCRβ-3:GCCCTATCCTGGGTCCACTCGTC(SEQ ID NO.39)

crRNA-TCRβ-4:AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.40)

CrRNA-dCK-1 is selected from following sequence：

crRNA-dCK-1:AACATTGCACCATCTGGCAACA(SEQ ID NO.41)

crRNA-dCK-2:TCAGCCAGCTCTGAGGGGACCCG(SEQ ID NO.42)

crRNA-dCK-3:AACATTGCACCATCTGGCAACAG(SEQ ID NO.43)

crRNA-dCK-4:TTGATGCGGGTCCCCTCAGAGCT(SEQ ID NO.44)

crRNA-dCK-5:CGGAGGCTCCTTACCGATGTTCC(SEQ ID NO.45)

crRNA-dCK-6:AGGATATTCACAAATGTTGACT(SEQ ID NO.46)

crRNA-dCK-7:TGAATATCCTTAAACAATTGTGTG(SEQ ID NO.47)

Preferably, use sgRNA and crRNA sequences used in CRISPR technologies for：

sgRNA-TCRα:AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10)

sgRNA-TCRβ:GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13)

sgRNA-dCK:TTCCTGAACCTGTTGCCAGA(SEQ ID NO.26)

crRNA-TCRα:ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.36)

crRNA-TCRβ:AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.40)

crRNA-dCK:TGAATATCCTTAAACAATTGTGTG(SEQ ID NO.47)。

Relative to prior art, advantages of the present invention is as follows,

It is double that the universal CART/TCRT cells with chemotherapeutic drug resistance of the present invention realize the progress of TCR and dCK genes Knock out, remain the targeting of tumour specific antigen, TCR knockout eliminates GvHD；DCK chemotherapeutics simultaneously Fludarabine and Clofarabine use removes the T cell in host, so as to prevent allogeneic T cells to be arranged Reprimand, and the allogeneic T cells that dCK molecules knock out are because resistant to Fludarabine and Clofarabine can deposit over a long time It is living to play anti-tumor activity；Can be as a kind of universal simple, low cost, high activity CART/TCRT cell preparations make With.

The present invention has the construction method of the universal CART/TCRT cells of chemotherapeutic drug resistance, uses Cas9 and Cpf1 Technology carries out double knockouts, caused double negative cells group, TCR negative cells to TCR the and dCK genes in CART/TCRT cells Reach 90%, dCK gene knockout levels and reach 70%；This cell mass loses GvHD activity and weakened to chemotherapeutics simultaneously Fludarabine and Clofarabine susceptibility；Sorted by the way that single TCR/CD3 is negative, TCR negative cells groups reach 99%.

Brief description of the drawings

The primary T cells surface TCR/CD3 compounds of Fig. 1 .Cas9 and Cpf1 mediation knock out；1a is situated between for CRISPR/Cas9 The TCR/CD3 compounds led knock out, and 1b is that the TCR/CD3 compounds of CRISPR/Cpf1 mediations knock out.

The CART cell TCR/dCK molecules of Fig. 2 .Cas9 and Cpf1 mediation are double to be knocked out；2a, which is that TCR/dCK molecules are double, to be knocked out carefully The TCR/CD3 of cellular surface compound expression, 2b, which is that the TCR/dCK molecules of Cas9 mediations are double, knocks out striking for intracellular dCK genes Except detection, 2c is the double knockout detections for knocking out intracellular dCK genes of the TCR/dCK molecules of Cpf1 mediations.

Fig. 3 .TCR/dCK are double to knock out CART/TCRT gene expression detections；3a, which is that TCR/dCK is double, knocks out CART gene expressions, 3b, which is that TCR/dCK is double, knocks out TCRT gene expressions.

Fig. 4 .TCR/dCK are double to knock out CART/TCRT cell cytokine secretions；4a, which is that TCR/dCK is double, knocks out CART cells Cytokine secretion, 4b, which is that TCR/dCK is double, knocks out TCRT cell cytokine secretions.

Fig. 5 .TCR/dCK are double to knock out the detection of CART/TCRT cell tumours killing activity；5a, which is that TCR/dCK is double, knocks out CART The anti-tumor activity detection of cell, 5b are the double anti-tumor activity detections for knocking out TCRT cells of TCR/dCK.

Fig. 6 are using different sgRNA and crRNA to T cell TCR α, TCR β and the editorial efficiency of dCK molecules.

Embodiment

Embodiment 1：

Primary T cells expand.Primary human T-Cells are being contained into 10%FBS, 100U/ml penicillin, 100 μ g/ml sulfate chains Mycin, cultivate in 10mM Hepes RPMI 1640, and using coupling CD3/CD28 antibody magnetic bead according to 1：3 ratio is entered Line activating stimulates.Cell is counted, every 2 days addition culture mediums, T is collected on appropriate opportunity by cell size and growth parameter(s) Cell, carry out functional examination or freezen protective.

CRISPR design and implementation

Sequence in sgRNA and crRNA targeting TCR α constant region exons 1, TCR β constant regions 1 and 2 exons 1s share Sequence, dCK exons 1 sequence, and be cloned into the carrier containing T7 promoters.These plasmid restriction enzymes are linear Change.Transcribed using MEGAscript T7Transcription kits (Life Technologies, Carlsbad, CA) SgRNA/crRNA, RNA is stored in -80 DEG C.

SgRNA the and crRNA sequences of Select to use of the present invention are as follows：

SgRNA-TCR α are selected from following sequence：

sgRNA-TCRα-1:GAGAATCAAAATCGGTGAAT(SEQ ID NO.1)

sgRNA-TCRα-2:GCTGGTACACGGCAGGGTCA(SEQ ID NO.2)

sgRNA-TCRα-3:GTCAGGGTTCTGGATATCTG(SEQ ID NO.3)

sgRNA-TCRα-4:ACAAAACTGTGCTAGACATG(SEQ ID NO.4)

sgRNA-TCRα-5:CTTCAAGAGCAACAGTGCTG(SEQ ID NO.5)

sgRNA-TCRα-6:TGGAATAATGCTGTTGTTGA(SEQ ID NO.6)

sgRNA-TCRα-7:TAGGCAGACAGACTTGTCAC(SEQ ID NO.7)

sgRNA-TCRα-8:TGGATTTAGAGTCTCTCAGC(SEQ ID NO.8)

sgRNA-TCRα-9:TCTCTCAGCTGGTACACGGC(SEQ ID NO.9)

sgRNA-TCRα-10:AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10)

SgRNA-TCR β are selected from following sequence：

sgRNA-TCRβ-1:GAGCAGCCGCCTGAGGGTCT(SEQ ID NO.11)

sgRNA-TCRβ-2:GAGCTGGTGGGTGAATGGGA(SEQ ID NO.12)

sgRNA-TCRβ-3:GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13)

sgRNA-TCRβ-4:GGCTCTCGGAGAATGACGAG(SEQ ID NO.14)

sgRNA-TCRβ-5:GGAGAATGACGAGTGGACCC(SEQ ID NO.15)

sgRNA-TCRβ-6:GGCGCTGACGATCTGGGTGA(SEQ ID NO.16)

sgRNA-TCRβ-7:GTTGCGGGGGTTCTGCCAGA(SEQ ID NO.17)

sgRNA-TCRβ-8:GCAGTATCTGGAGTCATTGA(SEQ ID NO.18)

sgRNA-TCRβ-9:GGCACACCAGTGTGGCCTTT(SEQ ID NO.19)

sgRNA-TCRβ-10:GGCTCAAACACAGCGACCTC(SEQ ID NO.20)

SgRNA-dCK is selected from following sequence：

sgRNA-dCK-1:GAACATCGGTAAGGAGCCTC(SEQ ID NO.21)

sgRNA-dCK-2:TTAAACAATTGTGTGAAGAT(SEQ ID NO.22)

sgRNA-dCK-3:ATTGTGTGAAGATTGGGAAG(SEQ ID NO.23)

sgRNA-dCK-4:ATCAAGAAAATCTCCATCGA(SEQ ID NO.24)

sgRNA-dCK-5:GTCTTTCTCAGCCAGCTCTG(SEQ ID NO.25)

sgRNA-dCK-6:TTCCTGAACCTGTTGCCAGA(SEQ ID NO.26)

sgRNA-dCK-7:GTAAGGAGCCTCCGGAAATG(SEQ ID NO.27)

CrRNA-TCR α are selected from following sequence：

crRNA-TCRα-1:GAGTCTCTCAGCTGGTACACGGC(SEQ ID NO.28)

crRNA-TCRα-2:CATGTGCAAACGCCTTCAACAAC(SEQ ID NO.29)

crRNA-TCRα-3:CACATGCAAAGTCAGATTTGTTG(SEQ ID NO.30)

crRNA-TCRα-4:TTGCTCCAGGCCACAGCACTGTT(SEQ ID NO.31)

crRNA-TCRα-5:TCTGTGATATACACATCAGAATC(SEQ ID NO.32)

crRNA-TCRα-6:TGACACATTTGTTTGAGAATCAA(SEQ ID NO.33)

crRNA-TCRα-7:TTTGAGAATCAAAATCGGTGAAT(SEQ ID NO.34)

crRNA-TCRα-8:AGAATCAAAATCGGTGAATAGGC(SEQ ID NO.35)

crRNA-TCRα-9:ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.36)

CrRNA-TCR β are selected from following sequence：

crRNA-TCRβ-1:GGTGTGGGAGATCTCTGCTTCTG(SEQ ID NO.37)

crRNA-TCRβ-2:CCTCGGTGTCCTACCAGCAAGGG(SEQ ID NO.38)

crRNA-TCRβ-3:GCCCTATCCTGGGTCCACTCGTC(SEQ ID NO.39)

crRNA-TCRβ-4:AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.40)

CrRNA-dCK-1 is selected from following sequence：

crRNA-dCK-1:AACATTGCACCATCTGGCAACA(SEQ ID NO.41)

crRNA-dCK-2:TCAGCCAGCTCTGAGGGGACCCG(SEQ ID NO.42)

crRNA-dCK-3:AACATTGCACCATCTGGCAACAG(SEQ ID NO.43)

crRNA-dCK-4:TTGATGCGGGTCCCCTCAGAGCT(SEQ ID NO.44)

crRNA-dCK-5:CGGAGGCTCCTTACCGATGTTCC(SEQ ID NO.45)

crRNA-dCK-6:AGGATATTCACAAATGTTGACT(SEQ ID NO.46)

crRNA-dCK-7:TGAATATCCTTAAACAATTGTGTG(SEQ ID NO.47)

Preferably, according to gene editing efficiency, use sgRNA the and crRNA sequences that CRISPR technologies preferably use for：

sgRNA-TCRα:AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10)

sgRNA-TCRβ:GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13)

sgRNA-dCK:TTCCTGAACCTGTTGCCAGA(SEQ ID NO.26)

crRNA-TCRα:ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.36)

crRNA-TCRβ:AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.40)

crRNA-dCK:TGAATATCCTTAAACAATTGTGTG(SEQ ID NO.47)。

The gene editing in T cell is realized using the method for electroporation

Gene editing in T cell is realized using the means of electroporation：First, T cell was at the 0th day, by being coupled CD3/ The magnetic bead of CD28 antibody enters line activating, and in the transfection that the 2nd~5 day carries out CRISPR, gene editing was collected at the 9th to the 11st day T cell；

For the gene editing of CART/TCRT cells, in addition to except the 1st day plus virus carried out CAR/TCR transfections to T cell, its He is same as above step.

The operating procedure of electroporation method approximately as：

1. using Opti-MEM cleaning T cells 3 times, each 300g, centrifuge 10 minutes and remove supernatant；

2. T cell is resuspended in Opti-MEM, make its final concentration of 10^6~10^8 cells/mL；

3. 20~100ug Cas9/Cpf1 albumen and 20~100ug sgRNA/crRNA mixed at room temperature are incubated 10 minutes；

4. albumen is mixed with sgRNA/crRNA mixtures with 100ul cell suspensions

5. carry out electricity using electroporation to turn.BTX-ECM830 parameter is 200~500V, 100us~3ms.(electroporation bag Include BTX, Biorad, Lonza etc.)

6. after electroporation, cell is immediately placed in 2mL pre-temperature culture mediums, and at 37 DEG C, 5%CO₂Culture or 32 DEG C, 5%CO₂Lower culture 1 day, then puts back to 37 DEG C, 5%CO₂Culture.

The gene knockout in primary T cells is realized by CRISPR

Because the TCR on allogeneic T cells can cause GvHD to react, while allogeneic T cells can be clear by host T cell identification Remove, present invention employs TCR the and dCK molecules for knocking out T cell surface simultaneously to prepare universal allogeneic T cells.TCR knockout Eliminate GvHD；The T that dCK chemotherapeutics Fludarabine and Clofarabine use are removed in host simultaneously is thin Born of the same parents, so as to prevent allogeneic T cells to be ostracised, and allogeneic T cells that dCK molecules knock out because to Fludarabine and Resistant can survive over a long time of Clofarabine plays anti-tumor activity.

We carry out gene knockout using CRISPR/Cas9 to TCR α, the β chain on allogeneic T cells first, find TCR α, β The knockout of chain destroys the formation of the TCR/CD3 complexs on T cell surface, and the editorial efficiency of TCR α, β chains respectively reaches 94.1% and 73.5% (Fig. 1).CRISPR/Cpf1 is a kind of new gene editing system, and its identification module (PAM) is different from Cas9 NGG, and it is TTTN, the analysis tool case of gene editing is expanded, possibility is provided for operation AT enriched DNA fragments. To realize the gene editing in primary T cells, we are cut using Cpf1 to TCR α, β chains, and it knocks out efficiency and reached respectively To 93% and 69.3% (Fig. 1).

Universal allosome CART/TCRT cells are built by CRISPR gene knockouts

In order to produce the T cell resistant to chemotherapeutics Fludarabine and Clofarabine, the present invention is simultaneously The response molecule for destroying its radiotherapy or chemotherapy to allogeneic T cells progress gene editing is realized logical with generating resistance T cell with this With the production of type T cell.

The present invention is simultaneously by combining virus transfection external source CAR/TCR genes and using Cas9/Cpf1 simultaneously thin to CART TCR and dCK genes in born of the same parents are operated, and realize the universal allosome CART/TCRT cell purposes of structure.

Double knockouts are carried out to TCR the and dCK genes in CART/TCRT cells using Cas9 and Cpf1, it is caused double-negative Cell mass, TCR negative cells reach 90%, dCK gene knockout levels and reach 70% (Fig. 2).This cell mass loses GvHD simultaneously Activity simultaneously weakens the susceptibility to chemotherapeutics Fludarabine and Clofarabine.Sorted by the way that single TCR/CD3 is negative, TCR negative cells groups reach 99%.Universal CART/TCRT cells can be used as.

Use flow cytomery T cell surface gene expression

The monoclonal antibody and reagent used.BD Biosciences (San Jose, CA)：APC-anti-CD3 (555335), FITC-anti-CD8 (555366), PE-anti-CD8 (555635), PE-anti-CD107 (555801), PE- Anti-B2m (551337), FITC-anti-HLA (555552)；Beckman Coulter (Pasadena, CA)：PE-anti- Vb13.1(IM2021U).Data are obtained using BD LSR2, and by FlowJo versions 7.6.1 (Tree Star, Inc.Ashland, OR) analysis.

Flow cytometer showed shows that T cell CAR and the TCR expression that TCR and dCK Gene Doubles knock out respectively reaches 84% He 91% (Fig. 3).

The function assessment research of universal CAR/TCRT cells

It is of the invention respectively to its targets neoplastic cells toxicity in order to verify the function of universal CAR/TCRT cells, and Tested in terms of cytokine secretion.

The double CAR/TCRT cells knocked out of TCR and dCK have the anti-tumor activity equal with wild type CAR/TCRT；And And the cell factor IL2 and IFNr (Fig. 4,5) of secretion peer-level.

CD107a staining procedures approximately as：In the RPMI culture mediums of 96 orifice plates, with effector cell：T cell ratio is 1：1 (1 × 10^5 effector cell:1 × 10^5 target) plating cells.CD107 antibody is added, and plate is incubated at 37 DEG C Educate 1 hour, then add Golgi stop, and plate is incubated in addition 2.5~3 hours.Then addition CD8 and CD3 antibody, and 37 DEG C incubate 30 minutes.After incubation terminates, cell is washed with FACS buffer solution, passes through flow cytometer showed.

Cell factor ELISA measure approximately as：In the complete medium of the RPMI 1640 containing 10% hyclone Middle washing target tumour cell, and suspended with 1 × 10^6 cell/ml.The 100 every kind of target cell types of μ l are added in duplicate In 96 hole round bottom plates.Washing effect T cell, and suspended in complete medium with 1 × 10^6 cell/ml, then will 100ul T cells combine with the target cell in designation hole.Plate is incubated 18 to 20 hours at 37 DEG C.

After incubation terminates, collect supernatant and carry out ELISA measure.

Cytotoxic T cell killing activity detection based on luciferase.By the tumour cell of targeted expression luciferase Wash in complete medium, and be resuspended with 1 × 10^5 cell/ml, then by 100ul tumour cells and the T of different proportion Cell (such as 30:1,15:1 etc.) it is incubated, is cultivated 18 to 20 hours at 37 DEG C together.After incubation terminates, substrate is added to carefully In born of the same parents and measure is luminous immediately, and result of calculation.

It should be noted that above-described embodiment is only presently preferred embodiments of the present invention, it is not used for limiting the present invention's Protection domain, the equivalent substitution or replacement made on the basis of the above belong to protection scope of the present invention.

Sequence table

<110>Nanjing Bei Heng bio tech ltd

<120>Universal CART/TCRT cells and its construction method with chemotherapeutic drug resistance

<160> 47

<170> SIPOSequenceListing 1.0

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<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

gagaatcaaa atcggtgaat 20

<210> 2

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

gctggtacac ggcagggtca 20

<210> 3

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

gtcagggttc tggatatctg 20

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

acaaaactgt gctagacatg 20

<210> 5

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

cttcaagagc aacagtgctg 20

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

tggaataatg ctgttgttga 20

<210> 7

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

taggcagaca gacttgtcac 20

<210> 8

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

tggatttaga gtctctcagc 20

<210> 9

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

tctctcagct ggtacacggc 20

<210> 10

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

agagtctctc agctggtaca 20

<210> 11

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

gagcagccgc ctgagggtct 20

<210> 12

<211> 20

<212> DNA

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<400> 12

gagctggtgg gtgaatggga 20

<210> 13

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<213>Artificial sequence (Artificial Sequence)

<400> 13

gctgtcaagt ccagttctac 20

<210> 14

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 14

ggctctcgga gaatgacgag 20

<210> 15

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 15

ggagaatgac gagtggaccc 20

<210> 16

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 16

ggcgctgacg atctgggtga 20

<210> 17

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 17

gttgcggggg ttctgccaga 20

<210> 18

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 18

gcagtatctg gagtcattga 20

<210> 19

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 19

ggcacaccag tgtggccttt 20

<210> 20

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 20

ggctcaaaca cagcgacctc 20

<210> 21

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 21

gaacatcggt aaggagcctc 20

<210> 22

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 22

ttaaacaatt gtgtgaagat 20

<210> 23

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 23

attgtgtgaa gattgggaag 20

<210> 24

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 24

atcaagaaaa tctccatcga 20

<210> 25

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 25

gtctttctca gccagctctg 20

<210> 26

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 26

ttcctgaacc tgttgccaga 20

<210> 27

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 27

gtaaggagcc tccggaaatg 20

<210> 28

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 28

gagtctctca gctggtacac ggc 23

<210> 29

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 29

catgtgcaaa cgccttcaac aac 23

<210> 30

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 30

cacatgcaaa gtcagatttg ttg 23

<210> 31

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 31

ttgctccagg ccacagcact gtt 23

<210> 32

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 32

tctgtgatat acacatcaga atc 23

<210> 33

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 33

tgacacattt gtttgagaat caa 23

<210> 34

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 34

tttgagaatc aaaatcggtg aat 23

<210> 35

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 35

agaatcaaaa tcggtgaata ggc 23

<210> 36

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 36

attctcaaac aaatgtgtca caa 23

<210> 37

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 37

ggtgtgggag atctctgctt ctg 23

<210> 38

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 38

cctcggtgtc ctaccagcaa ggg 23

<210> 39

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 39

gccctatcct gggtccactc gtc 23

<210> 40

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 40

agccatcaga agcagagatc tcc 23

<210> 41

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 41

aacattgcac catctggcaa ca 22

<210> 42

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 42

tcagccagct ctgaggggac ccg 23

<210> 43

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 43

aacattgcac catctggcaa cag 23

<210> 44

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 44

ttgatgcggg tcccctcaga gct 23

<210> 45

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 45

cggaggctcc ttaccgatgt tcc 23

<210> 46

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 46

aggatattca caaatgttga ct 22

<210> 47

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 47

tgaatatcct taaacaattg tgtg 24

Claims

1. the universal CART/TCRT cells with chemotherapeutic drug resistance, it is characterised in that the universal CART/TCRT is thin Born of the same parents are the allogeneic T cells for having Chimeric antigen receptor and φt cell receptor, φt cell receptor α, β chain and deoxycytidine kinase point Son is knocked.

2. universal CART/TCRT cells as claimed in claim 1, it is characterised in that the Chimeric antigen receptor is targeting CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, AFP, Alpha folate Receptor, CEA, PSCA, PSMA, Her2, EGFR, IL13R α 2, GD2, NKG2D, EGFRvIII, CS1, BCMA, Mesothelin Chimeric antigen receptor.

3. universal CART/TCRT cells as claimed in claim 1, it is characterised in that the φt cell receptor is targeting HBV, HPV E6, NY-ESO, mNY-ESO, WT1, MART-1, MAGE-A3, MAGE-A4, P53, Thyroglobulin, Tyrosinase φt cell receptor.

4. the construction method of the universal CART/TCRT cells with chemotherapeutic drug resistance, its feature as described in claim 1-3 It is, the knockout technology of φt cell receptor α, β chain and deoxycytidine kinase enzyme molecule is CRISPR technologies.

5. construction method as claimed in claim 4, it is characterised in that using sgRNA and crRNA used in CRISPR technologies Sequence is：

SgRNA-TCR α are selected from following sequence：

sgRNA-TCRα-1:GAGAATCAAAATCGGTGAAT(SEQ ID NO.1)

sgRNA-TCRα-2:GCTGGTACACGGCAGGGTCA(SEQ ID NO.2)

sgRNA-TCRα-3:GTCAGGGTTCTGGATATCTG(SEQ ID NO.3)

sgRNA-TCRα-4:ACAAAACTGTGCTAGACATG(SEQ ID NO.4)

sgRNA-TCRα-5:CTTCAAGAGCAACAGTGCTG(SEQ ID NO.5)

sgRNA-TCRα-6:TGGAATAATGCTGTTGTTGA(SEQ ID NO.6)

sgRNA-TCRα-7:TAGGCAGACAGACTTGTCAC(SEQ ID NO.7)

sgRNA-TCRα-8:TGGATTTAGAGTCTCTCAGC(SEQ ID NO.8)

sgRNA-TCRα-9:TCTCTCAGCTGGTACACGGC(SEQ ID NO.9)

sgRNA-TCRα-10:AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10)；

SgRNA-TCR β are selected from following sequence：

sgRNA-TCRβ-1:GAGCAGCCGCCTGAGGGTCT(SEQ ID NO.11)

sgRNA-TCRβ-2:GAGCTGGTGGGTGAATGGGA(SEQ ID NO.12)

sgRNA-TCRβ-3:GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13)

sgRNA-TCRβ-4:GGCTCTCGGAGAATGACGAG(SEQ ID NO.14)

sgRNA-TCRβ-5:GGAGAATGACGAGTGGACCC(SEQ ID NO.15)

sgRNA-TCRβ-6:GGCGCTGACGATCTGGGTGA(SEQ ID NO.16)

sgRNA-TCRβ-7:GTTGCGGGGGTTCTGCCAGA(SEQ ID NO.17)

sgRNA-TCRβ-8:GCAGTATCTGGAGTCATTGA(SEQ ID NO.18)

sgRNA-TCRβ-9:GGCACACCAGTGTGGCCTTT(SEQ ID NO.19)

sgRNA-TCRβ-10:GGCTCAAACACAGCGACCTC(SEQ ID NO.20)；

SgRNA-dCK is selected from following sequence：

sgRNA-dCK-1:GAACATCGGTAAGGAGCCTC(SEQ ID NO.21)

sgRNA-dCK-2:TTAAACAATTGTGTGAAGAT(SEQ ID NO.22)

sgRNA-dCK-3:ATTGTGTGAAGATTGGGAAG(SEQ ID NO.23)

sgRNA-dCK-4:ATCAAGAAAATCTCCATCGA(SEQ ID NO.24)

sgRNA-dCK-5:GTCTTTCTCAGCCAGCTCTG(SEQ ID NO.25)

sgRNA-dCK-6:TTCCTGAACCTGTTGCCAGA(SEQ ID NO.26)

sgRNA-dCK-7:GTAAGGAGCCTCCGGAAATG(SEQ ID NO.27)；

CrRNA-TCR α are selected from following sequence：

crRNA-TCRα-1:GAGTCTCTCAGCTGGTACACGGC(SEQ ID NO.28)

crRNA-TCRα-2:CATGTGCAAACGCCTTCAACAAC(SEQ ID NO.29)

crRNA-TCRα-3:CACATGCAAAGTCAGATTTGTTG(SEQ ID NO.30)

crRNA-TCRα-4:TTGCTCCAGGCCACAGCACTGTT(SEQ ID NO.31)

crRNA-TCRα-5:TCTGTGATATACACATCAGAATC(SEQ ID NO.32)

crRNA-TCRα-6:TGACACATTTGTTTGAGAATCAA(SEQ ID NO.33)

crRNA-TCRα-7:TTTGAGAATCAAAATCGGTGAAT(SEQ ID NO.34)

crRNA-TCRα-8:AGAATCAAAATCGGTGAATAGGC(SEQ ID NO.35)

crRNA-TCRα-9:ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.36)；

CrRNA-TCR β are selected from following sequence：

crRNA-TCRβ-1:GGTGTGGGAGATCTCTGCTTCTG(SEQ ID NO.37)

crRNA-TCRβ-2:CCTCGGTGTCCTACCAGCAAGGG(SEQ ID NO.38)

crRNA-TCRβ-3:GCCCTATCCTGGGTCCACTCGTC(SEQ ID NO.39)

crRNA-TCRβ-4:AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.40)；

CrRNA-dCK-1 is selected from following sequence：

crRNA-dCK-1:AACATTGCACCATCTGGCAACA(SEQ ID NO.41)

crRNA-dCK-2:TCAGCCAGCTCTGAGGGGACCCG(SEQ ID NO.42)

crRNA-dCK-3:AACATTGCACCATCTGGCAACAG(SEQ ID NO.43)

crRNA-dCK-4:TTGATGCGGGTCCCCTCAGAGCT(SEQ ID NO.44)

crRNA-dCK-5:CGGAGGCTCCTTACCGATGTTCC(SEQ ID NO.45)

crRNA-dCK-6:AGGATATTCACAAATGTTGACT(SEQ ID NO.46)

crRNA-dCK-7:TGAATATCCTTAAACAATTGTGTG(SEQ ID NO.47)。

6. construction method as claimed in claim 4, it is characterised in that using sgRNA and crRNA used in CRISPR technologies Sequence is：

sgRNA-TCRα:AGAGTCTCTCAGCTGGTACA(SEQ ID NO.10)

sgRNA-TCRβ:GCTGTCAAGTCCAGTTCTAC(SEQ ID NO.13)

sgRNA-dCK:TTCCTGAACCTGTTGCCAGA(SEQ ID NO.26)

crRNA-TCRα:ATTCTCAAACAAATGTGTCACAA(SEQ ID NO.36)

crRNA-TCRβ:AGCCATCAGAAGCAGAGATCTCC(SEQ ID NO.40)

crRNA-dCK:TGAATATCCTTAAACAATTGTGTG(SEQ ID NO.47)。