title: Genome editing ofClostridium autoethanogenumusing CRISPR/Cas9.
abstract: BACKGROUND
  Impactful greenhouse gas emissions abatement can now be achieved through gas fermentation using acetogenic microbes for the production of low-carbon fuels and chemicals. However, compared to traditional hosts likeEscherichia colior yeast, only basic genetic tools exist for gas-fermenting acetogens. To advance the process, a robust genetic engineering platform for acetogens is essential.
  
  
  RESULTS
  In this study, we report scarless genome editing of an industrially used model acetogen,Clostridium autoethanogenum, using the CRISPR/Cas9 system. Initial efforts to retrofit the CRISPR/Cas9 system forC. autoethanogenumresulted in poor efficiency likely due to uncontrolled expression of Cas9. To address this, we constructed and screened a small library of tetracycline-inducible promoters that can also be used to fine-tune gene expression. With a new inducible promoter, the efficiency of CRISPR/Cas9-mediated desired gene deletion inC. autoethanogenumwas improved to over 50 %, making it a viable tool for engineeringC. autoethanogenum.
  
  
  CONCLUSIONS
  Addition of both an inducible promoter library and a scarless genome editing tool is an important expansion to the genetic tool box of industrialC. autoethanogenumstrain.
authors: LanzaTech Inc, 8045 Lamon Ave, Suite 400, Skokie, IL 60077 USA.
authors: Shilpa
authors: 0000-0003-3114-6056
authors: S
authors: Nagaraju
authors: Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.
authors: Naomi Kathleen
authors: NK
authors: Davies
authors: University of Massachusetts, Amherst, MA USA.
authors: David Jeffrey Fraser
authors: DJ
authors: Walker
authors: LanzaTech Inc, 8045 Lamon Ave, Suite 400, Skokie, IL 60077 USA.
authors: Michael
authors: M
authors: Köpke
authors: LanzaTech Inc, 8045 Lamon Ave, Suite 400, Skokie, IL 60077 USA.
authors: Séan Dennis
authors: SD
authors: Simpson
keywords: Acetogen; CRISPR/Cas9; Clostridium autoethanogenum; Gas fermentation; Genome editing; Inducible promoter