Method for screening biallelic mutant cell lines by Cas12a protein

A cell line and allelic technology, applied in the medical and biological fields, can solve the problems of inability to distinguish monoallelic mutations and biallelic knockout strains, time-consuming, labor-intensive, time- and money-increasing costs, etc. The effect of reducing the time and money cost of screening

Inactive Publication Date: 2019-11-12
THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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Problems solved by technology

This method is time-consuming and labor-intensive, the results are unstable, and it is impossible to distinguish between monoallelic mut

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  • Method for screening biallelic mutant cell lines by Cas12a protein
  • Method for screening biallelic mutant cell lines by Cas12a protein
  • Method for screening biallelic mutant cell lines by Cas12a protein

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Embodiment Construction

[0024] In order to express the present invention more clearly, the present invention will be further described below in conjunction with the accompanying drawings.

[0025] see figure 1 , the Cas12a / Cpf1 protein used in this application also belongs to the CRISPR-Cas family and has an endonuclease function similar to Cas9. At the same time, the combination of Cas12a-crRNA complex with complementary single-stranded DNA or double-stranded DNA activates its powerful Non-specific ssDNA trans-cleavage activity, labeling ssDNA with a fluorescence quenching (FQ) reporter group can be developed into a nucleic acid detection tool. When Cas12a-crRNA matches the nucleic acid to be detected, Cas12a cuts ssDNA-fluorescent quenching reporter group If the Cas12a-crRNA does not match or does not completely match the nucleic acid to be detected, Cas12a does not cut or partially cuts the ssDNA-fluorescence quenching reporter group, and there is no fluorescence or weak fluorescence.

[0026] se...

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Abstract

The invention relates to a method for screening biallelic mutant cell lines by Cas12a protein, is mainly used for screening biallelic mutant cell lines after CRISPR/Cas9 editing and solves the technical problem that biallelic mutant cell lines are difficult to screen out. A nucleic acid detection technology developed on the basis of Cas12a protein (including all bacterial sources) can screen biallelic mutant positive cells after CRISPR/Cas9 editing simply, efficiently and stably, and the method can partially replace a sequencing method and greatly reduces the screening cost.

Description

technical field [0001] The invention relates to the fields of medicine and biology, and relates to a method for screening biallelic mutant cell lines based on CRISPR-Cas9 system gene editing by using Cas12a protein. Background technique [0002] CRISPR / Cas9 is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be used to fight against invading viruses and foreign DNA. The CRISPR / Cas9 system provides immunity by incorporating fragments of invading foreign DNA into CRISPR and using corresponding CRISPR RNAs (crRNAs) to direct the degradation of homologous sequences. After modification by scientists, CRISPR / Cas9 is widely used in gene editing of bacteria, animals, plants, and mammalian cells. The gene mutant animals constructed by this method have a significantly higher germline transfer ability than traditional methods, and are a reliable, efficient and rapid new method for constructing knockout animal models, showing great pot...

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Application Information

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IPC IPC(8): C12N15/90C12N9/22G01N21/64
CPCC12N9/22C12N15/907G01N21/6428G01N2021/6432
Inventor 张国良肖国辉刘淑燕贺星张苏欧敏
Owner THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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