文献类型：期刊文献

英文题名：Immediate, multiplexed and sequential genome engineering facilitated by CRISPR/Cas9 in Saccharomyces cerevisiae

作者：Li, Zhen-Hai[1];Meng, Hao[2];Ma, Bin[1];Tao, Xinyi[1];Liu, Min[1];Wang, Feng-Qing[1];Wei, Dong-Zhi[1]

机构：[1]East China Univ Sci & Technol, Newworld Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Hunan Norchem Pharmaceut Co Ltd, Changsha, Peoples R China

年份：2020

卷号：47

期号：1

起止页码：83

外文期刊名：JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY

收录：;EI(收录号：20242616405998);WOS:【SCI-EXPANDED(收录号:WOS:000511920800007)】；

语种：英文

外文关键词：Saccharomyces cerevisiae; CRISPR; Cas9; Gene targeting; Cell factory

摘要：A method called Cas-3P allowing for immediate, multiplexed and sequential genome engineering was developed using one plasmid expressing Cas9 and three marked plasmid backbones (P1, P2 and P3) for guide RNA (gRNA) expression. The three marked gRNA plasmid backbones were recurred in a P1-P2-P3 order for sequential gene targeting, without construction of any additional plasmid and elimination of gRNA plasmid by induction in each round. The efficiency of direct gRNA plasmid curing mediated by Cas-3P was more than 40% in sequential gene targeting. Besides, Cas-3P allowed single-, double- and triple-loci gene targeting with an efficiency of 75%, 36.8% and 8.2% within 3-4 days, respectively. Through three sequential rounds of gene targeting within 10 days, S. cerevisiae was optimized for the production of patchoulol by replacing one promoter, overexpressing three genes and disrupting four genes. The work is important for practical application in the cell factory engineering of S. cerevisiae.