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

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• 作者：He Huang ; Changsheng Chai ; Ning Li ; Pete Rowe ; Nigel P. Minton ; Sheng Yang ; Weihong Jiang ; Yang Gu
• 刊名：ACS Synthetic Biology
• 出版年：2016
• 出版时间：December 16, 2016
• 年：2016
• 卷：5
• 期：12
• 页码：1355-1361
• 全文大小：383K
• ISSN：2161-5063

文摘

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 (P_thl and P_araE) 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.