Efficient and precise genome editing and gene transcription repression in Clostridium beijerinckii using CRISPR/Cas9 system

Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated (Cas) nuclease system has been recently explored as a powerful tool for genome engineering and gene expression regulation for many eukaryotic organisms. However, its utilization in bacteria is not very effective due to the limited non-homologous end joining and recombineering capabilities of bacteria. Here, we explored Streptococcus pyogenes CRISPR/Cas9 for genome editing and gene transcription repression in Clostridium beijerinckii, an industrial significant microorganism which is notorious for being difficult to metabolically engineer. We demonstrated that the constitutive expression of CIRSPR/dead Cas9 (dCas9) targeting on amylase gene promoter region could led to efficient repression of amylase activity. By combing inducible expression of Cas9 and plasmid-borne editing templates, we successfully achieved chromosomal gene deletion and DNA integration with high efficiency in a single step. We further achieved single nucleotide modification by applying innovative two-step approaches, which does not rely on the availability of Protospacer Adjacent Motif (PAM) sequences at or in the vicinity of the target locus and to our best knowledge this is the first time that such strategies have been reported. We noticed that severe vector integration event (VIE) could occur during the genome engineering process. We successfully employed CRISPR/Cas9 as an efficient tool for removing VIE mutants and selecting desirable ‘clean’ ones. The approached we developed here is broadly applicable and will open the way for precise genome editing in diverse microorganisms.