CRISPR-Cas9-driven genome editing in Bacillus methanolicus MGA3

<i>Bacillus methanolicus</i> is a thermophilic methylotroph that uses methanol as a sustainable feedstock in biotechnological industry. Here, we developed the CRISPR-Cas9 genome editing tool for <i>B. methanolicus</i> 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 <i>katA</i> and <i>ald</i> 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 <i>katA</i> locus was replaced with <i>mcherry</i>, and successful integration was verified by PCR and increased mCherry fluorescence relative to the wild type. The system was further used to delete <i>spo0A</i> and replace it with <i>mrpf1</i>. 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 <i>B. methanolicus</i>.