Method for site-specific integration of large DNA fragment in mammalian cell through CRISPR/Cas9

Abstract

The invention discloses a method for site-specific integration of a large DNA fragment in a mammalian cell through CRISPR / Cas9. The method comprises the following steps: (1) constructing a CRISPR / Cas9 targeting system of a target CCR5 gene, wherein the targeting system comprises a Cas9 / sgRNA expression vector and a targeting donor, the targeting donor comprises a promotor, a fluorescence labelling gene and a design, and the design of the targeting donor comprises a gRNA target sequence at the middle part and shares a homologous sequence with a target gene; (2) enabling the CRISPR / Cas9 targeting system to cotransfer the mammalian cell, separating the cell stably expressing fluorescence after cotransfecting, and conducting single cell clone culture; (3) detecting the site-specific integration efficiency through single cell clone PCR, and acquiring the efficient site-specific site-detected integration mammalian cell. The method can realize the site-specific site-detected integration of the large DNA fragment more than 6 kb in the mammalian cell.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for realizing site-specific integration of large fragments of DNA in mammalian cells by using CRISPR / Cas9. Background technique [0002] The site-directed integration of large fragments of foreign genes in mammalian cell genomes usually adopts the method of double crossover homologous recombination. The implementation steps of this technology are to construct a targeting donor containing two homology arms on both sides of the target gene , using two homologous arms to perform double-crossover homologous recombination with the target gene, thereby replacing the target gene with an exogenous DNA fragment between the two homologous arms. However, this technology has the following disadvantages: the efficiency of double-exchange homologous recombination is often low, generally 10 -6 ; Using this technology to achieve site-specific integration of foreign DNA frag...

AI Technical Summary

Problems solved by technology

However, this technology has the following disadvantages: the efficiency of double-exchange homologous recombination is often low, generally 10 -6 ; The exogenous DNA fragments that use this technology to achieve site-specific integration are relatively small, generally less than 2kb

The specific reason may be that the greater the physical distance between the two homology arms, the less favorable the occurrence of simultaneous homologous recombination exchange on both sides of the target gene.

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