One-step Multi-lociintegration Based On CRISPR-Cas9

Saccharomyces cerevisiae is one of model organisms commonly used in microbiology and molecular biology research due to advantages including simple culture method,short growth cycles and high genetic stability.Gene editing in Saccharomyces cerevisiae is the basis of metabolic engineering and synthetic biology research,including gene knockout,expression enhancement or degradation,insertion and integration of heterologous genes and so on.Multi-loci integration is an important technology in metabolic engineering research.Heterologous expression of biosynthetic pathways is usually regulated by multiple genes.Efficient and convenient multi-loci gene editing method can significantly improve experimental efficiency,reduce time cost and promote research progress.There are two crucial factors that affect the efficiency of multi-loci integration,homologous arm length and Rad51.Generally,the homologous recombination efficiency can be improved by optimizing the homologous arm length,and the homologous recombination repair ability of yeast can also be improved by increasing the expression level of Rad51 protein.In addition,this paper found and proved for the first time that Brex27,a short peptide of 36 nt encoded on exon 27 of C-terminal of BRCA2 protein from human,could bind to Rad51 protein of yeast and enrich it in the neighborhood of Brex27.In order that Rad51 is enriched near the DSB cleaved by Cas9 to improve the efficiency of homologous recombination,we constructed the Cas9-Brex27 fusion protein.this project based on the CRISPR-Cas9 gene editing system and developed a multi-site editing method,realized the one-step multi-loci integration of heterologous expression pathway of four genes.We optimized the length of homologous arm to 100 bp with the Cas9-Brex27 fusion protein,and the four-loci integration efficiency was increased to 78%,which is the highest level of multi-loci integration at present.