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Gene editing system for preparing allotransplantable T cells

A technology of allotransplantation and gene editing, applied in plant gene improvement, genetic engineering, introduction of foreign genetic material using vectors, etc., can solve the problems of long cycle, high cost and complex construction process.

Active Publication Date: 2019-04-16
广东赤萌医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because ZFN or TALEN technology belongs to the first and second generation gene editing technology, there are great limitations in application, such as complex construction process, long cycle, high cost, etc.

Method used

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  • Gene editing system for preparing allotransplantable T cells
  • Gene editing system for preparing allotransplantable T cells
  • Gene editing system for preparing allotransplantable T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 gRNA preliminary screening

[0047] 1. Preparation of gRNA targeting TCRA gene

[0048] (1) The gRNA sequence designed according to the sequence of the TCRA gene (including the constant region of the α chain and the β chain):

[0049] The target sequence corresponding to the gRNA of the CRISPR / SpCas9 system is shown in the table below:

[0050] Table 1 Target sequence corresponding to the gRNA of the CRISPR / SpCas9 system for the TCRA gene.

[0051]

[0052]

[0053] The target sequence corresponding to the gRNA of the CRISPR / SaCas9 system is shown in the table below:

[0054] Table 2 The target sequence corresponding to the gRNA of the CRISPR / SaCas9 system for the TCRA gene.

[0055]

[0056]

[0057] (2) Synthesize the sense strand and antisense strand of the target sequence corresponding to the gRNA respectively (add cacc at the 5'-end of the sense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, then add cac...

Embodiment 2

[0098] Example 2 Screening for gRNA combinations of TCRA genes

[0099] In Example 1, through T7E1 enzyme digestion and agarose electrophoresis analysis, the gRNA with higher mutation efficiency screened out was combined to perform mutation operation on the TCRA gene. The specific steps are as follows:

[0100] 1. The gRNA combination is shown in the table below:

[0101] Table 4 The target sequence corresponding to the gRNA combination of the CRISPR / SaCas9 system for the TCRA gene:

[0102]

[0103] The plasmids prepared in 3(6) of Example 1 were co-transfected into HEK293T cells according to the above combination method, and the transfection method was referred to 4(1)-4(3) of Example 1;

[0104] 48 hours after transfection, amplify and purify the target fragment containing the target site according to the method of 5(1)~5(4) in Example 1;

[0105] Using DL2000 (TAKARA) as a reference, the length of the above PCR product was detected by 2% agarose gel electrophoresis. ...

Embodiment 3

[0111] Example 3 CRISPR / Cas9-mediated site-directed homologous recombination of TCRA gene

[0112] The gRNA or gRNA combination with higher mutation efficiency screened in Example 1 or Example 2 is used for CRISPR / Cas9-mediated integration and insertion of a foreign DNA sequence at a specific site in the TCRA gene (Cas9 cleavage site) . The following examples take gRNA target sequence SEQ ID NO: 3 as an example, and similarly can demonstrate the implementation effect of other gRNA or gRNA combinations.

[0113] 1. Homologous recombination donor vector construction

[0114] (1) Use the HEK293T cell genome as a template, use SEQ ID NO: 65 and SEQ ID NO: 66 as primers to amplify the upstream homology arm, and use SEQ ID NO: 67 and SEQ ID NO: 68 as primers to amplify the downstream homology arm , The PCR product was detected by 1% agarose gel electrophoresis, and the target fragment was recovered by cutting the gel;

[0115] Table 6 Amplification primers 65, 66, 67, 68 of the u...

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Abstract

The invention provides a gene editing system for preparing allotransplantable T cells. The system is characterized by comprising sgRNA, wherein the sgRNA in a CRISPR / Cas9 specific knockout human TCR gene is used for specifically targeting a TCR gene. The target sequence of the sgRNA on the TCR gene conforms to a sequence arrangement rule of 5'-G(21N)NNGRRT-3 or 5'-ARRCNN(21N)C-3', the target sequence of the targeted knockout TCR gene takes virus as a transfection vector, and a chimeric antigen receptor gene with TCR gene homologous arms at the 5' end and the 3' end can transfect cells togetherwith the virus containing the target sequence of the TCR gene by using the virus as a vector when in use, or packs the target sequence of the TCR gene, Cas9 and the chimeric antigen receptor gene with the TCR gene homologous arms at the 5' end and the 3' end together in baculovirus and transfects cells with the baculovirus as a vector.

Description

technical field [0001] The present invention relates to the field of targeted gene editing, in particular to a technology for preparing allotransplantable T cells using gene editing technology, specifically a gene editing system for preparing allotransplantable T cells. Background technique [0002] In recent years, the use of immunotherapy to treat tumors has received extensive attention. Among the many immunotherapies, the therapy based on Chimeric Antigen Receptor T cells (CAR-T) has the most remarkable and mature effect. At present, many clinical trials have proved that this technology can cure a variety of tumors, such as B cell tumors. The basic principle is to use genetic engineering methods to integrate the receptor gene that can specifically recognize tumor cells, and use the integrative virus as a carrier to integrate the receptor gene into the genome of the patient's T cells, so that the transformed T cells have The purpose of specifically recognizing and killin...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12N15/866
CPCC12N9/22C12N15/86C12N15/907C12N2710/14043
Inventor 祝海宝黄雨亭陈梓珊罗思施陶米林梁福才
Owner 广东赤萌医疗科技有限公司
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