Cell enrichment technology with C.T base substitution using inactive screening agent resistance gene as reporting system and application of technology

A technology of resistance gene and screening agent, applied in genetic engineering, recombinant DNA technology, introduction of foreign genetic material using vectors, etc.
CN110628794AActive Publication Date: 2019-12-31BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
Publication Date
2019-12-31

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Abstract

The invention discloses a cell enrichment technology with C.T base substitution using an inactive screening agent resistance gene as a reporting system and application of the technology. A vector of the cell enrichment technology comprises the following reagents: sgRNA, a C.T base substitution system and a screening agent resistance gene with loss of function. The sgRNA is composed of tRNA-sgRNA targeting a target sequence of an object gene and tRNA-sgRNA targeting a target sequence of the screening agent resistance gene with loss of function. The function of the screening agent resistance gene with loss of function can be recovered by performing C.T base substitution on the target sequence of the screening agent resistance gene with loss of function, by the C.T base substitution system, under the guide of tRNA-sgRNA targeting the target sequence of the screening agent resistance gene with loss of function. According to the present invention, cell enrichment with C.T base substitutionat the cell level is realized, and the efficiency of C.T base substitution is greatly improved.
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Description

technical field

[0001] The invention relates to the field of biotechnology, in particular to a C·T base replacement cell enrichment technology using an inactivated screening agent resistance gene as a reporter system and its application. Background technique

[0002] CRISPR-Cas9 technology has become a powerful genome editing method and has been widely used in many tissues and cells. The CRISPR / Cas9 protein-RNA complex is positioned on the target by the guide RNA (guide RNA), cuts and generates a DNA double-strand break (dsDNA break, DSB), and then the organism will instinctively initiate a DNA repair mechanism to repair the DSB. There are generally two repair mechanisms, one is non-homologous end joining (NHEJ), and the other is homologous recombination (homology-directed repair, HDR). Usually NHEJ accounts for the majority, so the random indels (insertions or deletions) generated by the repair are much higher than the precise repair. For precise base substitution, the ap...

Claims

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