title: CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii.
abstract: 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.
authors.affiliation: 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.
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.affiliation: Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, University of Nottingham, Nottingham, UK.
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Minton
authors.affiliation: Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, University of Nottingham, Nottingham, UK.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Wen
authors.affiliation: 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.
authors.forename: Jinle
authors.initials: J
authors.lastname: Liu
authors.affiliation: 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.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Yang
authors.affiliation: 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.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: 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.
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Ren
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Yang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.|Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. syang@sibs.ac.cn.|Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China. syang@sibs.ac.cn.|Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing, China. syang@sibs.ac.cn.
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems; D046969:Clostridium acetobutylicum / Q000235:genetics*; D046970:Clostridium beijerinckii / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D007218:Industrial Microbiology; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D003850:Deoxyribonuclease I
keywords: CRISPR-Cas9; Clostridium; Gene expression; Genome editing; Nickase