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

A construction method and a technology for repairing a vector, applied in the field of genetic engineering, can solve the problems of comprehensive research on gene restriction, inability to introduce foreign genes at a fixed point, etc., and achieve the effect of high assembly efficiency

Pending Publication Date: 2018-04-20
GUANGDONG UNIV OF PETROCHEMICAL TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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.

Method used

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

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Embodiment Construction

[0035] The present invention will be described in further detail below through examples, which are only used to illustrate the present invention and do not limit the scope of the present invention.

[0036] The hypocotyls of aseptic seedlings of Eucalyptus vulgaris were used as explants for tissue culture regeneration. The specific methods are as follows:

[0037] 1 Cultivation of Arabidopsis seedlings

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/82
CPCC12N15/66C12N15/8213
Inventor 欧阳乐军李莉梅徐锡荣刘雅丽柳镜炬梁裕华
Owner GUANGDONG UNIV OF PETROCHEMICAL TECH
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