Method for multicopy integration of target gene to saccharomyces cerevisiae genome

Abstract

A method for multicopy integration of a target gene to a saccharomyces cerevisiae genome includes: selecting target sequences, designing homologous arm primers in length of 15-90bp on two sides of each cleavage site in the corresponding target sequences to primer front ends of amplification target gene cassettes to synthesize homologous arm loaded primers, and amplifying the target gene cassettesto obtain donor DNA fragments; subjecting transcribed gRNA vectors in corresponding target sequences and the donor DNA fragments to co-transformation into Cas9 nuclease expressing saccharomyces cerevisiae to realize gene integration; removing Cas9 nuclease expression vectors and the transcribed gRNA vectors by a non-resistant flat plate. The method has advantages that the homologous arm primers ontwo sides of each cleavage site is in sequence length of 15-90bp, and by respective adding of the homologous arm primers to upstream and downstream primers of the target gene cassettes, an integratorgene template can be obtained directly through once PCR, so that multi-copy integration of target genes is realized. Compared with an existing method, the method has advantages of low time consumption and simplicity in operation.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a method for integrating multiple copies of a target gene into the genome of Saccharomyces cerevisiae. Background technique [0002] The Saccharomyces cerevisiae system is one of the most important exogenous gene expression systems and has been widely used in the synthesis of biological agents. Saccharomyces cerevisiae has an efficient homologous recombination mechanism, as long as there is a 30-50bp homologous segment between two DNA molecules, homologous recombination can be carried out accurately and effectively. According to the characteristics of Saccharomyces cerevisiae that is prone to homologous recombination, using repetitive sequences as homologous recombination sites of integration vectors is an effective way to increase the copy number of exogenous genes in the yeast genome. [0003] The rDNA in the yeast genome has 100-200 repeating units, and the copy number has a sig...

AI Technical Summary

Problems solved by technology

[0007] In the current method of high-copy integration of the target gene using CRISPR / Cas9 technology, the homology arms of the donor DNA are mostly 500bp or even more than 1000bp, and it is necessary to PCR amplify the upper and lower homology arms and the gene expression cassette to be inserted separately. They are assembled, the steps are cumbersome

In addition, when integrating multiple genes at a time, too long homology arms will affect the selection of target sequences, making it difficult to integrate more genes at one time

Images