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ACS Synth Biol. 2016 Dec 16;5(12):1355-1361. Epub 2016 Jun 15.

CRISPR/Cas9-Based Efficient Genome Editing in Clostridium ljungdahlii, an Autotrophic Gas-Fermenting Bacterium.

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Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai 200032, China.
Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, University of Nottingham , Nottingham, NG7 2RD, U.K.
Jiangsu National Synergetic Innovation Center for Advanced Materials, SICAM , 200 North Zhongshan Road, Nanjing 210009, China.
Shanghai Collaborative Innovation Center for Biomanufacturing Technology , 130 Meilong Road, Shanghai 200237, China.


Acetogenic bacteria have the potential to convert single carbon gases (CO and CO2) into a range of bulk chemicals and fuels. Realization of their full potential is being impeded by the absence of effective genetic tools for high throughput genome modification. Here we report the development of a highly efficient CRISPR/Cas9 system for rapid genome editing of Clostridium ljungdahlii, a paradigm for the commercial production of ethanol from synthesis gas. Following the experimental selection of two promoters (Pthl and ParaE) for expression of cas9 and the requisite single guide RNA (sgRNA), the efficiency of system was tested by making precise deletions of four genes, pta, adhE1, ctf and pyrE. Deletion efficiencies were 100%, >75%, 100% and >50%, respectively. The system overcomes the deficiencies of currently available tools (more rapid, no added antibiotic resistance gene, scarless and minimal polar effects) and will find utility in other acetogens, including the pathogen Clostridium difficile.


CRISPR/Cas9; Clostridium ljungdahlii; autotrophic bacterium; gas fermentation; rapid genome editing; strong promoters

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