CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9-system-based Saccharomyces cerevisiae genome concurrent multiplex edition vector and application thereof

Abstract

The invention relates to a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9-system-based Saccharomyces cerevisiae genome concurrent multiplex edition vector and application thereof. Cas9 protein expression cassettes and a plurality of sgRNA (small guide ribonucleic acid) scaffold expression cassettes are integrated into the vector to obtain a vector-Cas9-sgRNA scaffold, and a plurality of sgRNA segments designed and synthesized by using ADE1 as a target are integrated into the vector-Cas9-sgRNA scaffold, thereby obtaining the Saccharomyces cerevisiae genome concurrent multiplex edition vector. By adopting a simple substance granule single-copy vector system, one plasmid is utilized to simultaneously express the Cas9 protein and three sgRNA scaffolds, so that three target genes can be edited simultaneously; and thus, the multigene edition operation is very simple. Besides, the target gene edition system only needs one-step component and conversion, and thus, is simple to operate.

Description

technical field [0001] The invention relates to a Saccharomyces cerevisiae genome parallel multiple editing vector based on a CRISPR-Cas9 system and an application thereof. Background technique [0002] With the completion of human genome sequencing, life science research has entered a post-genome era aimed at revealing gene functions, and genome editing technology has become an important research tool and means. Traditional genome editing technology uses homologous recombination mechanism (homologous recombination) to edit genes in a targeted manner, which can help researchers clarify the function of genes. Traditional genome editing technology uses long fragments of DNA as target sites, and the "positioning system" must also It is a long DNA fragment, the construction of the editing system is complex and time-consuming, the experiment cycle is long, the efficiency is very low, and the gene is prone to mutation and other defects. The second-generation genome editing techno...

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Problems solved by technology

Traditional genome editing technology uses homologous recombination mechanism (homologous recombination) to edit genes in a targeted manner, which can help researchers clarify the function of genes. Traditional genome editing technology uses long fragments of DNA as target sites, and the "positioning system" must also It is a long DNA fragment, the construction of the editing system is complex and time-consuming, the experiment cycle is long, the efficiency is very low, and the gene is easy to mutate and other defects

The second-generation genome editing technology (including ZFN system and TALEN system) has solved the problem of low specificity of the editing system to a certain extent, but still failed to solve the outstanding problems of cumbersome and time-consuming construction of the editing system

[0013] The existing CRISPR-Cas9 system consists of two plasmids, one of which expresses the Cas9 protein with the activity of cutting double-stranded DNA. If three genes are to be edited, the other three plasmids are required to express three sgRNAs. These four Plasmids require four different screening markers, and the compatibility of plasmids must also be considered. The construction of such a genome editing system is very difficult and cumbersome, which greatly prolongs the experimental cycle

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