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

•Customized CRISPR/Cas9 genome engineering tool has been developed for C. beijerinckii.•Clean markerless gene deletion from the chromosome has been achieved through a single-step transformation.•High efficiency of gene deletion has been achieved based on the CRISPR/Cas9 assisted homologous recombination event.•This report represents the first application of the CRISPR/Cas9 system for genome engineering in Clostridium.•The protocols and principles developed in this study provided valuable references for genome engineering in other microorganisms.

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.