Method for screening biallelic mutant cell lines by Cas12a protein

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...

AI Technical Summary

Problems solved by technology

This method is time-consuming and labor-intensive, the results are unstable, and it is impossible to distinguish between monoallelic mutations and biallelic knockout strains. It must be verified by subsequent sequencing, which greatly increases the cost of time and money.

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