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 draw‐back for the commercial use of Cas9 is the IP landscape requiring a licence 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, USA). 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.