gRNA for knockout of wild type T cell TCR [beta] strand and method

A wild-type, cell technology, applied in the field of gene knockout, can solve the problems of unstable RNA, low transfection efficiency, complicated preparation, etc., and achieve the effect of high gene knockout efficiency and simple preparation method.

Active Publication Date: 2017-11-17
THE FIFTH AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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Problems solved by technology

However, the above technologies also have many shortcomings in practical applications. Among them, RNAi technology only reduces the expression of wild-type alpha and beta chains at the RNA level, and it is an incomplete knockout, and the remaining RNA can continue to express TCR protein molecules ; ZFN technology requires the preparation of multiple vectors, which can be knocked out only after transcribed into RNA in vitro and then transferred into T cells. The RNA is unstable and cumbersome to prepare; Low, affecting the knockout efficiency of TCR wild-type alpha and beta chains

Method used

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  • gRNA for knockout of wild type T cell TCR [beta] strand and method
  • gRNA for knockout of wild type T cell TCR [beta] strand and method
  • gRNA for knockout of wild type T cell TCR [beta] strand and method

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Embodiment

[0030] A method for knocking out the wild-type TCR beta chain, comprising the following steps:

[0031] 1) Design of gRNA target site, synthesis of DNA sequence encoding gRNA;

[0032] The sequence information of the human TCR beta chain constant coding region (TRBC) gene obtained through THE INTERNATIONAL IMMUNOGENETICS INFORMATIONSYSTE is shown in SEQ ID NO:4.

[0033] >M12887|TRBC1*01|Homo sapiens|F|EX1+EX2+EX3+EX4|213..599+1041..1058+1211..1317+1640..1657|531nt|1|+1|||| 531+0=531|||

[0034] Put the sequence information of human TRBC on the online CRISPR / Cas9 design tool crispr gRNA designtool (https: / / www.atum.bio / eCommerce / cas9 / input) for computer prediction to obtain several DNA coding sequences of gRNA targeting TRBC. Among them, the nucleic acid sequence of the designed gRNA is shown in SEQ ID NO: 1, the gRNA is obtained by adding CACC to the 5' end of its corresponding DNA sequence to obtain the forward nucleotide sequence, and adding AAAC to the 5' end of its comp...

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Abstract

The invention discloses gRNA for knockout of a wild type T cell TCR [beta] strand and a method. The sequence of the gRNA is as shown in SEQ ID NO: 1, by utilizing a CRISPR / Cas9 technology, the gRNA and CRISPR / Cas9 jointly infect a T cell, the wild type T cell TCR [beta] strand is knocked out, and the T cell lacking the wild type TCR [beta] strand is constructed and used for CAR-T or TCR-T cellular immunotherapy. According to the gRNA for knockout of the wild type T cell TCR [beta] strand and the method, the knockout efficiency is high, the preparation method is relatively simple and easy, and T cells lacking wild type TCR [beta] strands can be provided for clinic rapidly and efficiently.

Description

technical field [0001] The invention relates to the technical field of gene knockout, in particular to a gRNA and a method for knocking out the TCR beta chain of wild-type T cells. Background technique [0002] Tumor is currently the biggest killer of human health. Existing treatments including surgery, chemotherapy, and radiotherapy cannot completely eradicate tumors. Immune T cell therapy has brought great hope for the recovery of tumor patients. At present, the methods of immune T cell therapy mainly include CAR-T and TCR-T cell technology. The principle of these T cell therapy is to transfect tumor-specific chimeric antigen receptor (CAR) or T cell receptor (TCR) gene into normal T cells. Above, make it acquire the ability of tumor-specific recognition, so as to kill tumor cells. However, since normal T cells contain their own T cell receptors (wild-type alpha and beta chains), these wild-type alpha and beta chains can affect the expression of CAR-T or TCR-T, resulting ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N5/10C12N15/90
CPCC07K14/7051C12N15/113C12N15/907C12N2310/10
Inventor 翁锦生
Owner THE FIFTH AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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