{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T07:51:06Z","timestamp":1769673066913,"version":"3.49.0"},"reference-count":76,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T00:00:00Z","timestamp":1769558400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"ERA CoBioTech","award":["327216"],"award-info":[{"award-number":["327216"]}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>\n                    <jats:italic>Bacillus methanolicus<\/jats:italic>\n                    is a thermophilic methylotroph that uses methanol as a sustainable feedstock in biotechnological industry. Here, we developed the CRISPR-Cas9 genome editing tool for\n                    <jats:italic>B. methanolicus<\/jats:italic>\n                    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\n                    <jats:italic>katA<\/jats:italic>\n                    and\n                    <jats:italic>ald<\/jats:italic>\n                    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\n                    <jats:italic>katA<\/jats:italic>\n                    locus was replaced with\n                    <jats:italic>mcherry<\/jats:italic>\n                    , and successful integration was verified by PCR and increased mCherry fluorescence relative to the wild type. The system was further used to delete\n                    <jats:italic>spo0A<\/jats:italic>\n                    and replace it with\n                    <jats:italic>mrpf1<\/jats:italic>\n                    . 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\n                    <jats:italic>B. methanolicus<\/jats:italic>\n                    .\n                  <\/jats:p>","DOI":"10.3389\/fmicb.2025.1728984","type":"journal-article","created":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T18:54:39Z","timestamp":1769626479000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["CRISPR-Cas9-driven genome editing in Bacillus methanolicus MGA3"],"prefix":"10.3389","volume":"16","author":[{"given":"May L. 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