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

Jul 29, 2025ACS synthetic biology

Editing and controlling multiple genes at once in Bacillus subtilis using CRISPR-MAD7

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Abstract

MAD7 enables multiplex genome editing of up to four loci in B. subtilis.

MAD7 is lethal when combined with chromosome-targeting gRNA, which facilitates robust counterselection.
A strain was engineered to carry the MAD7-gRNA machinery in a conditionally inactivated state.
Both MAD7 and its inactive variant (dMAD7) can be regulated by inactivation at elevated temperatures.
The MAD7-gRNA complex allows for simultaneous deletion, mutation, or insertion of multiple genes.
A strategy was established to remove MAD7 and the gRNA alongside desired genome edits.

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With the advent of MAD7, a Cpf1-like nuclease, there has been a renewed focus on the development of CRISPR-based genome engineering tools in recent years. To improve genome engineering methodologies in, we revisited the potential of MAD7 for gene modification and expression interference. A key challenge in these endeavors is the limited transformation efficiency. To overcome this, we developed an efficient transformation protocol using strains overexpressing competence genes. Our results showed that although MAD7 together with achromosome-targeting gRNA is lethal, enabling robust counterselection, we successfully engineered a strain carrying the MAD7-gRNA machinery in a reversibly inactivated state, marking a significant advance in the field. We demonstrated that both MAD7 and its catalytically inactive variant (dMAD7) can be conditionally regulated by inactivation at elevated temperatures. In addition, the MAD7-gRNA complex is effective for multiplex genome editing, allowing for the simultaneous deletion, mutation, or insertion of up to four loci, and enabling the combination of gene deletion, gene insertion, and point mutations. Furthermore, we established a strategy that achieves the simultaneous removal of MAD7 and the gRNA along with the desired genome edits. Altogether, this comprehensive study underscores the versatility of MAD7 for complex, scarless genome engineering and lays a strong foundation for further advancing genetic manipulation in. B. subtilis B. subtilis B. subtilis

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

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