Method for verifying feasibility of CRISPR-Cas9 system for knocking out candida utilis target gene

Abstract

The invention relates to a method for verifying feasibility of a CRISPR-Cas9 system for knocking out a candida utilis target gene. The method comprises the steps of S1, determining the multiple of a candida utilis chromosome; S2, constructing a candida utilis expression vector having a Cas9 segment; S3, constructing an sgRNA preliminary plasmid containing a tRNAGlu gene and an sgRNA downstream expression unit; S3, assigning a sequence to be knocked out and a target point sequence, adding a sticky end at the target point and a complementary sequence 5' end, performing annealing to obtain dual chains, and enabling the dual chains and the preliminary plasmid to be combined into an sgRNA mature recombinant plasmid; S5, constructing a recombinant donor fragment containing a knockout sequence; S6, transforming the sgRNA mature recombinant plasmid into candida utilis having a Cas9 fragment; and S7, transforming the mature recombinant plasmid and a donor into candida utilis carrying Cas9, detecting knockout, or performing knockout and introducing gene expression changes or phenotype changes of an exogenous gene, and verifying whether Cas9 mediated candida utilis gene knockout is feasible or not.

Description

technical field [0001] The present invention relates to the field of gene knockout, and more specifically, relates to a method for verifying the feasibility of knocking out the target gene of Candida utilis by CRISPR-Cas9 system. Background technique [0002] Since CRISPR-Cas9 technology was reported, it has been successfully applied to gene knockout research in various organisms, including animals, plants, bacteria, haploid or diploid yeast. Candida utilis is a polyploid yeast with no sexual generation and no haploid strains. Therefore, the gene knockout operation of Candida utilis is currently carried out, and the Cre-loxP recombinase system is usually used to genetically modify it. This method first uses the homologous recombination mechanism to knock out an allele of polyploid yeast, and then Use the Cre-loxP system to remove the antibiotic resistance gene, and then knock out the other allele; repeat the steps of knocking out the allele and removing the antibiotic resis...

AI Technical Summary

Problems solved by technology

[0003] The above-mentioned gene knockout process for Candida utilis is cumbersome and complicated, so constructing a set of simple, convenient and efficient knockout system in Candida utilis is very important for the gene function and metabolic engineering transformation of Candida utilis. very important

However, it is still unknown whether the CRISPR-Cas9 system, hereinafter referred to as the Cas9 system, can be applied to the gene knockout of Candida utilis. Therefore, the CRISPR-Cas9 system has not been applied to the gene knockout of Candida utilis. Unable to construct a Candida utilis gene knockout system mediated by the Cas9 system

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