Expanding and understanding the CRISPR toolbox for Bacillus subtilis with MAD7 and dMAD7

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. Development of a CRISPR method with the commercially attractive nuclease, MAD7, and confirmation of its comparable efficiency to Cas9 for the counterselection of unmodified Bacillus subtilis cells. Engineering of a dMAD7 variant for single and multiplexed knock‐down of gene expression by CRISPRi.