Multiplex Genome Editing and Regulation in Bacillus subtilis with CRISPR-MAD7

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• CRISPR-based genome engineering tools are rapidly evolving.
• MAD7, a Cpf1-like nuclease, offers new possibilities for genetic manipulation.
• Improving genome engineering efficiency in B. subtilis is crucial for research.

Purpose of the Study:

• To explore the utility of the MAD7 system for gene modification and expression interference in B. subtilis.
• To develop an efficient transformation protocol to overcome low transformation efficiency.
• To establish a robust and versatile genome engineering platform in B. subtilis.

Main Methods:

• Developed an efficient transformation protocol by overexpressing competence genes in B. subtilis.
• Engineered a strain with a reversibly inactivated MAD7-gRNA system for conditional control.
• Utilized temperature-sensitive inactivation for both active MAD7 and catalytically inactive dMAD7 variants.
• Demonstrated multiplex genome editing capabilities for simultaneous modification of multiple loci.
• Established a strategy for simultaneous removal of the MAD7-gRNA machinery and genome edits.

Main Results:

• MAD7 with a B. subtilis targeting gRNA proved lethal, enabling effective counterselection.
• Successfully created a strain with a reversibly inactivated MAD7-gRNA system.
• Demonstrated conditional regulation of MAD7 and dMAD7 activity at elevated temperatures.
• Achieved multiplex genome editing, modifying up to four loci simultaneously (deletions, insertions, point mutations).
• Successfully removed the MAD7-gRNA components along with the desired genetic alterations.

Conclusions:

• MAD7 is a versatile tool for complex, scarless genome engineering in B. subtilis.
• The developed system enhances genetic manipulation capabilities in this bacterium.
• This study provides a strong foundation for advanced genetic engineering applications in B. subtilis.