A RecET-assisted CRISPR–Cas9 genome editing in Corynebacterium glutamicum

作者：Bo Wang, Qitiao Hu, Yu Zhang, Yu Zhang, Ruilin Shi, Xin Chai, Zhe Liu, Yun Zhang, Yun Zhang, Yun Zhang, Tingyi Wen · 发表于：Microbial Cell Factories · 年份：2018 · DOI：10.1186/s12934-018-0910-2 · 被引用次数：87 · 研究领域：Microbial Metabolic Engineering and Bioproduction、CRISPR and Genetic Engineering、Plant biochemistry and biosynthesis

BACKGROUND: Extensive modification of genome is an efficient manner to regulate the metabolic network for producing target metabolites or non-native products using Corynebacterium glutamicum as a cell factory. Genome editing approaches by means of homologous recombination and counter-selection markers are laborious and time consuming due to multiple round manipulations and low editing efficiencies. The current two-plasmid-based CRISPR-Cas9 editing methods generate false positives due to the potential instability of Cas9 on the plasmid, and require a high transformation efficiency for co-occurrence of two plasmids transformation. RESULTS: Here, we developed a RecET-assisted CRISPR-Cas9 genome editing method using a chromosome-borne Cas9-RecET and a single plasmid harboring sgRNA and repair templates. The inducible expression of chromosomal RecET promoted the frequencies of homologous recombination, and increased the efficiency for gene deletion. Due to the high transformation efficiency of a single plasmid, this method enabled 10- and 20-kb region deletion, 2.5-, 5.7- and 7.5-kb expression cassette insertion and precise site-specific mutation, suggesting a versatility of this method. Deletion of argR and farR regulators as well as site-directed mutation of argB and pgi genes generated the mutant capable of accumulating L-arginine, indicating the stability of chromosome-borne Cas9 for iterative genome editing. Using this method, the model-predicted target genes were modified to...