Saccharomyces cerevisiae genome editing carrier based on CRISPR-Cas9 system and application of carrier

Abstract

The invention relates to a saccharomyces cerevisiae genome editing carrier based on a CRISPR-Cas9 system and application of the carrier. According to the saccharomyces cerevisiae genome editing carrier, a Cas9 protein expression cassette and an sgRNA scaffold expression cassette are integrated in the carrier, and a carrier-Cas9-sgRNA scaffold is obtained; any one of multiple sgRNA fragments which are designed and synthesized by taking ADE1 as a target site is integrated in the carrier-Cas9-sgRNA scaffold, and the saccharomyces cerevisiae genome editing carrier is obtained. According to the saccharomyces cerevisiae genome editing carrier based on the CRISPR-Cas9 system and the application of the carrier, a single plasmid and single copy carrier system is adopted, one plasmid is adopted to express Cas9 protein and sgRNA scaffold simultaneously, construction and conversion of a target gene editing system only need one step, and operation is easy and convenient.

Description

technical field [0001] The invention relates to a Saccharomyces cerevisiae genome 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 technology (including ZF...

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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

Since the CRISPR-Cas9 system uses a two-plasmid system, it is difficult to construct, and the two-plasmid is easily affected by plasmid compatibility

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