Human iPS cell gene editing and screening method

A cell gene and screening method technology, applied in the field of gene editing and screening of human iPS cells, can solve the problems of expensive flow sorting instruments, high experimental conditions, and the unknown influence of human genome, etc., and achieve the convenience of single cloning Effects of selection and establishment of lines and improvement of gene editing rate
CN112266935APending Publication Date: 2021-01-26UNION STEMCELL & GENE ENG
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
UNION STEMCELL & GENE ENG
Publication Date
2021-01-26

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Abstract

The invention discloses a human iPS cell gene editing and screening method. The method comprises the following steps: introducing editing plasmids into iPS cells by an electroporation method; producing gene-edited cells through resistance screening and enrichment; and performing low-density passage, and selecting monoclonal cell colonies for enlarged culture and identification. An EF1 alpha promoter is used for promoting free plasmids capable of simultaneously promoting Cas9 and screening resistance protein expression in human iPS cells, after the plasmids enter the human iPS cells, Cas9 protein and resistance protein can be transiently and stably expressed in the cells at the same time, and in the most vigorous time period of plasmid transcriptional expression, screened substances are added into a culture environment. The iPS cells which obtain exogenous plasmids and express the resistance protein survive, besides, the expressed Cas9 protein is guided by sgRNA to be subjected to double-strand cleavage at the target sequence position of a genome for gene editing, and the cells which do not obtain the exogenous plasmids or are not expressed by the plasmids die due to the fact that the cells do not contain resistance. Use of a flow cytometry or insertion of exogenous genes into the cell genome is avoided.
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Description

technical field

[0001] The present invention relates to a method for gene editing and screening of cells, especially a method for gene editing and screening of human iPS cells, specifically, a method for gene knockout mediated by CRISPR / Cas9 and enrichment by resistance screening , and then the method of cell selection. 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 gene editing technology is a technology for specific DNA modification of targeted genes, and this technology is also a cutting-edge method for gene editing. The Cas9 enzyme protein can generate double-strand breaks at specific positions in the genome under the guidance of gRNA, thereby stimulating the DNA damage repair mechanism in cells. There are two main mechanisms for intracellular repair of DNA double-strand breaks: one is the non-homolo...

Claims

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