Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium
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ABSTRACT
The 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 genus
Clostridium
has lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied the
Streptococcus pyogenes
type II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing in
Clostridium acetobutylicum
DSM792. 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 in
C. acetobutylicum
. Subsequently, we targeted the carbon catabolite repression (CCR) system of
C. acetobutylicum
through transcriptional repression of the
hprK
gene 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 in
Clostridium pasteurianum
ATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes.
IMPORTANCE
After recognizing the industrial potential of
Clostridium
for 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 in
Clostridium acetobutylicum
, which is arguably the most common industrial clostridial strain. The developed genetic tools enable simpler, more reliable, and more extensive derivation of
C. acetobutylicum
mutant strains for industrial purposes. Similar approaches were also demonstrated in
Clostridium 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.
