Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium Academic Article uri icon

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  • ABSTRACTThe discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genusClostridiumhas lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied theStreptococcus pyogenestype II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing inClostridium acetobutylicumDSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression inC. acetobutylicum. Subsequently, we targeted the carbon catabolite repression (CCR) system ofC. acetobutylicumthrough transcriptional repression of thehprKgene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated inClostridium pasteurianumATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes.IMPORTANCEAfter recognizing the industrial potential ofClostridiumfor decades, methods for the genetic manipulation of these anaerobic bacteria are still underdeveloped. This study reports the implementation of CRISPR-Cas technology for genome editing and transcriptional regulation inClostridium acetobutylicum, which is arguably the most common industrial clostridial strain. The developed genetic tools enable simpler, more reliable, and more extensive derivation ofC. acetobutylicummutant strains for industrial purposes. Similar approaches were also demonstrated inClostridium pasteurianum, another clostridial strain that is capable of utilizing glycerol as the carbon source for butanol fermentation, and therefore can be arguably applied in other clostridial strains.


  • Bruder, Mark R
  • Pyne, Michael E
  • Moo-Young, Murray
  • Chung, Duane
  • Chou, C Perry

publication date

  • October 15, 2016