文献类型：期刊文献

英文题名：CRISPR-Cas12a assisted precise genome editing of Mycolicibacterium neoaurum

作者：Liu, Ke[1];Gao, Yang[1];Zhen-Hai Li[1];Liu, Min[1];Feng-Qing Wang[1];Dong-Zhi Wei[1]

机构：[1]East China Univ Sci & Technol, Newworld Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2022

卷号：66

起止页码：61

外文期刊名：NEW BIOTECHNOLOGY

收录：;EI(收录号：20214311053685);WOS:【SCI-EXPANDED(收录号:WOS:000731397200008)】；

基金：This research was financially supported by the National Natural Science Foundation of China (No. 21776075) , the Natural Science Foundation of Shanghai (No. 20ZR1415100) , and the National Key Research and Development Program of China (No. SQ2020YFC210061) .

语种：英文

外文关键词：CRISPR-Cas12a; Genome editing; Mycolicibacterium neoaurum; NHEJ; Gene integration

摘要：Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum, including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.