CRISPR-Cas9-driven genome editing in Bacillus methanolicus MGA3

Bacillus methanolicus is a thermophilic methylotroph that uses methanol as a sustainable feedstock in biotechnological industry. Here, we developed the CRISPR-Cas9 genome editing tool for B. methanolicus MGA3. This one-plasmid system induces Cas9-mediated double-strand breaks and exploits native DNA repair: homologous recombination for scarless deletions and gene replacements, and error-prone end-joining repair for mutagenesis in the absence of a repair template. Consistent with end-joining activity, Cas9 cutting without a template resulted in reproducible small indels near the cleavage site. Using homology-directed repair, we deleted the katA and ald genes, confirmed the edits by genome sequencing, and demonstrated the expected loss of catalase and alanine dehydrogenase activities, respectively; both phenotypes were restored by complementation. For targeted gene insertion, the katA locus was replaced with mcherry , and successful integration was verified by PCR and increased mCherry fluorescence relative to the wild type. The system was further used to delete spo0A and replace it with mrpf1 . The overall genome-editing efficiency exceeded 85%. This study demonstrates that the developed genome editing platform enables precise and efficient genetic modifications for metabolic engineering in B. methanolicus .