Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System

N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for was evaluated in by targeting and , two essential genes for acetate and butyrate production, respectively. and single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is