Li Qi, Chen Jun, Minton Nigel P, Zhang Ying, Wen Zhiqiang, Liu Jinle, Yang Haifeng, Zeng Zhe, Ren Xiaodan, Yang Junjie, Gu Yang, Jiang Weihong, Jiang Yu, Yang Sheng
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Graduate University of the Chinese Academy of Sciences, Beijing, China.
Biotechnol J. 2016 Jul;11(7):961-72. doi: 10.1002/biot.201600053. Epub 2016 Jun 13.
Solventogenic clostridia are important industrial microorganisms that produce various chemicals and fuels. Effective genetic tools would facilitate physiological studies aimed both at improving our understanding of metabolism and optimizing solvent productivity through metabolic engineering. Here we have developed an all-in-one, CRISPR-based genome editing plasmid, pNICKclos, that can be used to achieve successive rounds of gene editing in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 with efficiencies varying from 6.7% to 100% and 18.8% to 100%, respectively. The plasmid specifies the requisite target-specific guide RNA, the gene encoding the Streptococcus pyogenes Cas9 nickase and the genome editing template encompassing the gene-specific homology arms. It can be used to create single target mutants within three days, with a further two days required for the curing of the pNICKclos plasmid ready for a second round of mutagenesis. A S. pyogenes dCas9-mediated gene regulation control system, pdCASclos, was also developed and used in a CRISPRi strategy to successfully repress the expression of spo0A in C. acetobutylicum and C. beijerinckii. The combined application of the established high efficiency CRISPR-Cas9 based genome editing and regulation control systems will greatly accelerate future progress in the understanding and manipulation of metabolism in solventogenic clostridia.
产溶剂梭菌是重要的工业微生物，可生产多种化学品和燃料。有效的遗传工具将有助于开展生理学研究，旨在增进我们对新陈代谢的理解，并通过代谢工程优化溶剂生产率。在此，我们开发了一种基于CRISPR的一体化基因组编辑质粒pNICKclos，它可用于在丙酮丁醇梭菌ATCC 824和拜氏梭菌NCIMB 8052中实现连续多轮基因编辑，效率分别为6.7%至100%和18.8%至100%。该质粒指定了必需的靶向特异性引导RNA、编码化脓性链球菌Cas9切口酶的基因以及包含基因特异性同源臂的基因组编辑模板。它可用于在三天内创建单靶点突变体，再用两天去除pNICKclos质粒，为第二轮诱变做好准备。还开发了一种化脓性链球菌dCas9介导的基因调控控制系统pdCASclos，并将其用于CRISPRi策略，以成功抑制丙酮丁醇梭菌和拜氏梭菌中spo0A的表达。已建立的基于CRISPR-Cas9的高效基因组编辑和调控控制系统的联合应用，将极大地加速未来在理解和操纵产溶剂梭菌新陈代谢方面的进展。
