CRISPR(clustered regulatory interspersed short palindromic repeat)系统因其高效、无痕、简便等特点已被广泛应用于生物基因编辑研究领域.就微生物基因编辑而言,该系统的研究主要集中于单倍体细胞的基因编辑操作.然而,对于遗传背景更为复杂的多倍体细胞来说,高效基因编辑系统的匮乏是制约工业菌株遗传改造及应用的关键因素.因此,以酿酒酵母二倍体工业菌株为研究对象,构建并系统优化了RNA Pol Ⅱ(Pol Ⅱ)和RNA Pol Ⅲ(Pol Ⅲ)两种启动子驱动sgRNA表达的CRISPR/Cas9 基因编辑体系,同时对转化条件进行优化提高多位点基因编辑效率.研究结果表明,Pol Ⅱ启动子可实现 3 对等位基因(即 6 个基因)一次性敲除,效率高达68%.随后也对目标基因多位点敲入进行了初步探索.该研究建立的酿酒酵母工业菌株CRISPR/Cas9 基因编辑系统为菌株快速遗传改造提供强有力的技术支撑.
Optimization of a CRISPR/Cas9 gene editing system for type Ⅱ promoter-mediated sgRNA expression in industrial strains of Saccharomyces cerevisiae
The CRISPR(clustered regulatory interspersed short palindromic repeat)system has been widely used in biological gene editing research due to its high efficiency,tracelessness and simplicity.As far as microbial gene editing is concerned,the research of this system mainly focuses on the gene editing operation of haploid cells.However,for polyploid cells,which have a more complex genetic background,the lack of efficient gene editing systems is a key factor limiting the genetic modification and application of industrial strains.Therefore,in this study,a CRISPR/Cas9 gene editing system was constructed and systematically optimized for sgRNA expression driven by two promoters,RNA Pol Ⅱ(PolⅡ)and RNA PolⅢ(PolⅢ),using a diploid industrial strain of Saccharomyces cerevisiae,and optimized the transformation conditions to improve the efficiency of multilocus gene editing.The results showed that the Pol Ⅱ promoter could achieve three pairs of alleles(six genes)to be knocked out at one time with an efficiency as high as 68%.Subsequently,preliminary explorations of multilocus knock-down of target genes were also carried out.The CRISPR/Cas gene editing system for industrial strains of S.cerevisiae established in this study provides strong technical support for rapid genetic modification of strains.
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