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Updated: Dec 28, 2025

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
Published on: September 2, 2021
Expanding and understanding the CRISPR toolbox for Bacillus subtilis with MAD7 and dMAD7
Marcus A Price1, Rita Cruz2, James Bryson3
1School of Biological Sciences, Institute of Quantitative Biology, Biochemistry, and Biotechnology, University of Edinburgh, Edinburgh, UK.
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The CRISPR-Cas9 system has become increasingly popular for genome engineering across all fields of biological research, including in the Gram-positive model organism Bacillus subtilis. A major drawback for the commercial use of Cas9 is the IP landscape requiring a license for its use, as well as reach-through royalties on the final product. Recently an alternative CRISPR nuclease, free to use for industrial R&D, MAD7 was released by Inscripta (CO). Here we report the first use of MAD7 for gene editing in B. subtilis, in which editing rates of 93% and 100% were established. Additionally, we engineer the first reported catalytically inactive MAD7 (dMAD7) variant (D877A, E962A, and D1213A) and demonstrate its utility for CRISPR interference (CRISPRi) at up to 71.3% reduction of expression at single and multiplexed target sites within B. subtilis. We also confirm the CRISPR-based editing mode of action in B. subtilis providing evidence that the nuclease-mediated DNA double-strand break acts as a counterselection mechanism after homologous recombination of the donor DNA.
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