Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification in Clostridium Beijerinckii as an Example

The application of the CRISPR-Cas9 system as a tool to genetically engineer bacteria represents an important improvement from previous methods, such as use of heterologous counter-selection markers, recombinases or the group II intron, all of which have limitations. Many industrially important bacteria are tough to work with due to the physiological characteristics they have adopted from their natural environment, such as thick cell walls, high GC content, spore formation and lack of sufficient chromosomal DNA repair mechanisms. This study demonstrates the use of a chimeric single RNA-guided Streptococcus pyogenes CRISPR-Cas9 system as a selection tool for desired mutants, from recombinants of a plasmid-borne editing template, in place of Cas9-directed repair, using Clostridium beijerinckii as an example. High efficiency of gene deletion, integration and single nucleotide modification was achieved. We further proposed and investigated an innovative two-step approach, which does not rely on direct availability of Protospacer Adjacent Motif sequences for genome editing. Based on these advances, additional studies using a similar strategy has been adapted and applied to other bacterial species and strains, supporting the potential for precise genome editing by this approach.