CRISPR-Cas9-driven genome editing in Bacillus methanolicus MGA3

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bacillus methanolicus is a thermophilic methylotroph utilized in industry for its ability to use methanol.
• Efficient genetic tools are crucial for metabolic engineering of industrial microorganisms.

Purpose of the Study:

• To develop and validate a CRISPR-Cas9 genome editing system for Bacillus methanolicus MGA3.
• To enable precise gene deletions, replacements, and mutagenesis in B. methanolicus.

Main Methods:

• A one-plasmid CRISPR-Cas9 system was engineered for B. methanolicus.
• Exploited native DNA repair pathways (homologous recombination and end-joining) for genome modifications.
• Utilized homology-directed repair for scarless gene deletions and replacements, and error-prone end-joining for mutagenesis.

Main Results:

• Successfully deleted katA and ald genes, with corresponding loss of enzyme activity.
• Confirmed gene edits via genome sequencing and phenotypic analysis.
• Achieved targeted gene insertion by replacing katA with mcherry, verified by PCR and fluorescence.
• Demonstrated high overall genome-editing efficiency exceeding 85%.

Conclusions:

• The developed CRISPR-Cas9 system provides a precise and efficient platform for genetic manipulation in B. methanolicus.
• This tool is valuable for advancing metabolic engineering strategies in thermophilic methylotrophs.
• The system facilitates scarless gene editing and targeted gene insertion for strain improvement.