Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable "clean" Mutant Selection in Clostridium beijerinckii as an Example

CRISPR-Cas9 has been demonstrated as a transformative genome engineering tool for many eukaryotic organisms; however, its utilization in bacteria remains limited and ineffective. Here we explored Streptococcus pyogenes CRISPR-Cas9 for genome editing in Clostridium beijerinckii (industrially significant but notorious for being difficult to metabolically engineer) as a representative attempt to explore CRISPR-Cas9 for genome editing in microorganisms that previously lacked sufficient genetic tools. By combining inducible expression of Cas9 and plasmid-borne editing templates, we successfully achieved gene deletion and integration with high efficiency in single steps. We further achieved single nucleotide modification by applying innovative two-step approaches, which do not rely on availability of Protospacer Adjacent Motif sequences. Severe vector integration events were observed during the genome engineering process, which is likely difficult to avoid but has never been reported by other researchers for the bacterial genome engineering based on homologous recombination with plasmid-borne editing templates. We then further successfully employed CRISPR-Cas9 as an efficient tool for selecting desirable "clean" mutants in this study. The approaches we developed are broadly applicable and will open the way for precise genome editing in diverse microorganisms.