Journal
ACS SYNTHETIC BIOLOGY
Volume 5, Issue 12, Pages 1355-1361Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.6b00044
Keywords
CRISPR/Cas9; rapid genome editing; strong promoters; Clostridium ljungdahlii; autotrophic bacterium; gas fermentation
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Funding
- National High-tech Research and Development Program of China [2015AA020202]
- Chinese Academy of Sciences [KSZD-EW-Z-017-2, KGZD-EW-606]
- Synthetic Biology China-UK Partnering Award (Utilizing Steel Mill Off-Gas for Chemical Commodity Production using Synthetic Biology) - BBSRC [BB/L01081X/1]
- Synthetic Biology China-UK Partnering Award (Utilizing Steel Mill Off-Gas for Chemical Commodity Production using Synthetic Biology) - Chinese Academy of Sciences
- National Natural Science Foundation of China [31370133, 31121001]
- Youth Innovation Promotion Association CAS
- BBSRC [BB/L01081X/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/L01081X/1, 1370661] Funding Source: researchfish
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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.
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