文献类型：期刊文献

英文题名：CRISPR-Cas Assisted Shotgun Mutagenesis Method for EvolutionaryGenome Engineering

作者：Zhao, Ming[1,2];Gao, Miaomiao[1];Xiong, Liangbin[1,3];Liu, Yongjun[1];Tao, Xinyi[1];Gao, Bei[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]Anhui Polytech Univ, Coll Biol & Food Engn, Anhui Engn Lab Ind Microbiol Mol Breeding, Wuhu 241000, Peoples R China;[3]Shanghai Univ Med & Hlth Sci, Shanghai Key Lab Mol Imaging, Shanghai 201318, Peoples R China

年份：2022

卷号：11

期号：5

起止页码：1958

外文期刊名：ACS SYNTHETIC BIOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000805370400024)】；

基金：This work was financially supported by the Natural Science Foundation of Shanghai (No. 20ZR1415100) and the Chinese Postdoctoral Science Foundation (No. 2020M671021).

语种：英文

外文关键词：CRISPR-mediated genome engineering; whole-genome mutagenesis; iterative evolution

摘要：Genome mutagenesis drives the evolution of organisms. Here, wedeveloped a CRISPR-Cas assisted random mutation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library andSpCas9-NG to randomly damage genomes in a controllable shotgunlike manner thatthen triggers diverse and abundant mutations via low-fidelity repair. As a proof ofprinciple, CARM was applied to evolve the capacity ofSaccharomyces cerevisiaeBY4741to produce beta-carotene. After seven rounds of iterative evolution over two months, a beta-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in beta-carotene production and 857 diverse genomic mutations that comprised indels,duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes ofstrain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition,CARM was applied to evolve industrially relevantS. cerevisiaeCEN.PK2-1C for S-adenosyl-L-methionine production, which wasincreased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypesthat could further be investigated by reverse engineering