Conditional gene knockout method constructed by using CRISPR/Cas9 system

Abstract

The invention relates to a conditional gene knockout method constructed by using a CRISPR / Cas9 system. LoxP-antibiotic-LoxP and FRT-antibiotic-FRT-LoxP sequence insertion sites are selected from leftand right sides of a to-be-knocked-out area respectively; left and right homologous arms are selected from upstream and downstream positions of to-be-inserted sites, amplification, purification, enzyme digestion, linking and conversion are performed, and left and right targeting plasmids are obtained; Cas9 plasmid, the left or right targeting plasmid of the knockout area and corresponding gRNA expression plasmids are sequentially or simultaneously introduced into cells, screening is performed with antibiotics after transfection, and target cells with left and right targeting sequences insertedare screened out simultaneously; 4-OHT and DOX are used for inducing Cre-LoxP and FLP-FRT medicated homologous recombination, so that loxp sites are introduced in left and right sides of the to-be-knocked-out area; 4-OHT is used for inducing Cre-LoxP medicated homologous recombination, and cloning of target fragment knockout is obtained.

Description

technical field [0001] The invention relates to the technical field of gene knockout, in particular to a gene conditional knockout method constructed using CRISPR / Cas9 and Cre-LoxP systems. Background technique [0002] Gene knockout technology is an irreplaceable part in the construction of transgenic animals. The expression of genes is specific in time and space, and gene editing such as knockout and replacement of key genes in the body often leads to the death of animal embryos and hinders the study of gene function. The emergence of the conditional knockout method has solved the above-mentioned problems, allowing the expression or deletion of the target gene to occur at a specific stage of animal development or in a specific tissue or organ. As a new generation of gene knockout technology, conditional gene knockout technology can realize the specific expression of genes in space and time, and has obvious advantages and broad application prospects compared with constitut...

AI Technical Summary

Problems solved by technology

Therefore, this step requires a lot of time and effort, and is the rate-limiting step for conditional knockout of genes using the Cre / loxP system

[0008] (2) Cre enzyme, as an exogenous recombinase expressed in mammals, has certain potential toxicity, which can cause problems such as abnormal cell proliferation, DNA mismatch and chromosome deletion

[0011] In short, the traditional recombinase-mediated gene conditional knockout, which uses gene recombination under natural conditions, has low recombination efficiency

The traditional Cre / loxP system inserts the loxP site into the genome, the homology arms are long, and the efficiency of homologous recombination is low, which greatly affects the efficiency of gene conditional knockout

Images