Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system

J Biotechnol. 2015 Apr 20;200:1-5. doi: 10.1016/j.jbiotec.2015.02.005. Epub 2015 Feb 11.


The anaerobic spore-forming, gram-positive, solventogenic clostridia are notorious for being difficult to genetically engineer. Based on CRISPR/Cas9 assisted homologous recombination, we demonstrated that clean markerless gene deletion from the chromosome can be easily achieved with a high efficiency through a single-step transformation in Clostridium beijerinckii NCIMB 8052, one of the most prominent strains for acetone, butanol and ethanol (ABE) production. This highly efficient genome engineering system can be further explored for multiplex genome engineering purposes. The protocols and principles developed in this study provided valuable references for genome engineering in other microorganisms lacking developed genetic engineering tools.

Keywords: Butanol; CRISPR/Cas9; Clostridium; Genome engineering; Homologous recombination.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Chromosomes, Bacterial
  • Clostridium beijerinckii / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Gene Deletion*
  • Genes, Bacterial
  • Genetic Engineering
  • Genome, Bacterial