The CRISPR toolbox for the gram-positive model bacterium Bacillus subtilis

Area of Science:

• Molecular Biology
• Synthetic Biology
• Microbial Engineering

Background:

• CRISPR technology offers versatile genome engineering capabilities.
• Bacillus subtilis is a key industrial microorganism and model system.
• CRISPR was first adapted to Bacillus subtilis in 2016, spurring rapid development.

Purpose of the Study:

• To review the evolution and expansion of the CRISPR toolbox in Bacillus subtilis.
• To highlight advancements in CRISPR-based genome engineering tools for B. subtilis.
• To discuss strain development driven by CRISPR technology in B. subtilis.

Main Methods:

• Review of literature on CRISPR applications in Bacillus subtilis since 2016.
• Compilation of CRISPR tools for genome editing (point mutations, insertions, deletions) and gene expression control (CRISPRi, CRISPRa).
• Analysis of multiplex CRISPR applications and limitations.

Main Results:

• A comprehensive CRISPR toolbox for B. subtilis has been established, including tools for various editing functions.
• CRISPR enables precise genome modifications up to 38 kb and gene expression modulation.
• Multiplex CRISPR targeting up to six loci for point mutations is successful, but limitations exist for other functions.

Conclusions:

• CRISPR engineering has led to improved protein and metabolite production in Bacillus subtilis strains.
• The ongoing development of the CRISPR toolbox continues to enhance B. subtilis as an industrial chassis.
• Further development of multiplex CRISPR tools is needed for broader applications in B. subtilis.