Stable knockout single plasmid vector with coordination of transposon and CRISPR/Cas9 system and application of stable knockout single plasmid vector

Abstract

The invention discloses a stable knockout single plasmid vector with coordination of transposon and a CRISPR / Cas9 system and application of stable knockout single plasmid vector and belongs to the field of gene engineering. The single plasmid vector is a double-stranded circular plasmid containing an IRDR-L-IRDR-R box, and the IRDR-L-IRDR-R box comprises an IRDR-L sequence, a promoter, a gRNA scaffold sequence, a Cas9 protein sequence, a resistance screening gene sequence and an IRDR-R sequence. The single plasmid vector provided by the invention can realize expression of sgRNA and a Cas9 protein only needing once construction and once transfection. A method is simple in process, is efficient and rapid, greatly simplifies a plasmid construction flow, shortens an experiment period and improves the working efficiency. The vector provided by the invention carries a transposase recognition sequence, does not use a virus, can conveniently, rapidly and safely establish a gene knockout stablecell line and is beneficial to screening stable cell lines by comprising the puromycin screening resistance.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a single plasmid vector, in particular to a stable knockout single plasmid vector of a transposon cooperating with a CRISPR / Cas9 system and an application thereof. Background technique [0002] Gene function verification is the forerunner of genetic engineering, including two main methods: gene overexpression and gene silencing. The CRISPR / Cas9 system is an emerging accurate and efficient gene knockout system, which is based on the bacterial adaptive immune system and uses two main components, sgRNA and Cas9. Among them, sgRNA contains a site-specific target sequence, which guides Cas9 nuclease to accurately bind to a specific position in the genome to cut and form a DNA double-strand break. The inaccuracy of organism damage repair will lead to gene mutation at the target site, resulting in loss of its function. . The conventional CRISPR / Cas9 system is constructed from two plasm...

AI Technical Summary

Problems solved by technology

Among them, sgRNA contains a site-specific target sequence, which guides Cas9 nuclease to accurately bind to a specific position in the genome to cut and form a DNA double-strand break. The inaccuracy of organism damage repair will lead to gene mutation at the target site, resulting in loss of its function.

The conventional CRISPR / Cas9 system is constructed from two plasmids, and the sgRNA and Cas9 protein are expressed separately in the two plasmid systems, which has the defects of cumbersome construction process, long time and low efficiency

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