Construction method of homologous repair vector based on CRISPR/Cas9 system

Abstract

The invention relates to a construction method of a homologous repair vector based on a CRISPR/Cas9 system, and belongs to the technical field of genetic engineering. The construction method comprisesthe following steps: with a plasmid PCBC and wild-type arabidopsis genome as templates, amplifying a target band AS-gRNA containing a target sequence and an AS homologous repair template segment by virtue of PCR, implementing electrophoresis and gel cutting, and recovering the target band; implementing enzyme digestion on a plasmid PHDE-mCH by virtue of Bsa1; assembling and linking a PHDE-mCh vector, which undergoes complete enzyme digestion, with the ASgRNA by virtue of homologous recombinase, so that a recombinant plasmid PHDE-ASgRNA is formed, implementing transformation and sequencing identification, then linking the PHDE-ASgRNA plasmid, which is correct in sequencing and is subjected to enzyme digestion by virtue of EcoR1, with AS homologous repair template segment homologous recombinase, and implementing transformation and sequencing identification, so that the PHDE-ASgRNA-AS homologous repair vector is constructed. According to the technique provided by the invention, the method for constructing the CRISPR/Cas9 system which has a site-specific editing function on target biology genome is actually implemented; the vector is constructed just by conducting PCR by one step, sothat the method is simple and easy to implement; and the method, which is unnecessary to purify or recover a digestion vector and a PCR product, is high in assembly efficiency.

Description

technical field [0001] The invention relates to a method for constructing a homologous repair vector based on a CRISPR / Cas9 system, in particular to a method for constructing a cDNA library to construct a plasmid vector for screening, and belongs to the technical field of genetic engineering. Background technique [0002] CRISPR / Cas9 genome-directed editing technology is a technology developed in recent years for targeted and precise modification of the genome. By introducing exogenous DNA into a specific site of the recipient cell's chromosome, the genome can be specifically modified to study the function of the gene. This technology can perform operations such as deletion, knock-in, and nucleotide correction of target sites in the genome. In 2013, scientists first applied CRISPR / Cas9 to knock out genes in human and mouse cell lines, and then it was successfully applied in model plants and other crops, and the modified CRISPR / Cas9 system also quickly It has been applied t...

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Problems solved by technology

[0003] So far, the use of CRISPR / Cas9 system to realize the targeted editing of the genome can only target the targeted knockout of endogenous genes, and cannot realize the fixed-point introduction of exogenous genes, which limits the comprehensive study of gene functions.

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