gRNA subjected to wild type T cell TCR alpha chain knockout and method

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

Inactive Publication Date: 2017-10-10
THE FIFTH AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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  • Abstract
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  • Application Information

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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 molecu

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  • gRNA subjected to wild type T cell TCR alpha chain knockout and method
  • gRNA subjected to wild type T cell TCR alpha chain knockout and method
  • gRNA subjected to wild type T cell TCR alpha chain knockout and method

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[0028] Example

[0029] A method for knocking out the TCR alpha chain of wild-type T cells includes the following steps:

[0030] 1) Design of gRNA target site, synthesize the DNA sequence encoding gRNA;

[0031] Through the International Immunology Information System (THE INTERNATIONAL IMMUNOGENETICS INFORMATIONSYSTE), the sequence information of the human TCR alpha chain constant coding region (TRAC) gene is shown in SEQ ID NO:4.

[0032] > X02883|TRAC*01|Homosapiens|F|EX1+EX2+EX3|273..545+2408..2452+3324..3427|423nt|1|+1|||||423+0=423|||

[0033] Put the sequence information of human TRAC on the online CRISPR / Cas9 design tool crispr gRNA designtool (https: / / www.atum.bio / eCommerce / cas9 / input) and perform computer prediction to obtain several DNA coding sequences for TRAC gRNA. Among them, the nucleic acid sequence of the designed gRNA is shown in SEQ ID NO: 1. The gRNA adds CACC to the 5'end of its corresponding DNA sequence to obtain a forward nucleotide sequence, and adds AAAC to t...

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Abstract

The invention discloses a gRNA subjected to wild type T cell TCR alpha chain knockout and a method. The sequence of the gRNA is shown as SEQ ID NO:1. By using a CRISPR/Cas 9 technology, the gRNA and the CRISPR/Cas9 perform co-infection on T cells; the wild type T cell TCR alpha chain knockout is performed; the T cells lack of wild type T cell TCR alpha chains are built; the gRNA can be used for CAR-T or TCR-T cellular immunity treatment. The gRNA has high knockout rate; the preparation method is relatively simple and easy; the T cells lack of wild type T cell TCR alpha chains can be fast and efficiently provided for clinics.

Description

technical field [0001] The invention relates to the technical field of gene knockout, in particular to a gRNA and method for knocking out TCR alpha 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 in CA...

Claims

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Application Information

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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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