A-to-G), but cannot produce base transversions. Here we present BEs that cause C-to-A transversions in E. coli and C-to-G transversions in mammalian cells. Our glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a Uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, creating an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. [AU: unclear how this results in a transversion. Can this be

Base editing technology based on Clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) is a recent addition to the family of CRISPR technologies. Compared with the traditional CRISPR/Cas9 technology, it does not rely on DNA double strand break and homologous recombination, and can realize gene inactivation and point mutation more quickly and simply. Herein, we first developed a base editing method for genome editing in Bacillus subtilis utilizing CRISPR/dCas9 (a fully nuclease-deficient mutant of Cas9 from S. pyogenes) and activation-induced cytidine deaminase (AID). This method achieved three and four loci simultaneous editing with editing efficiency up to 100% and 50%, respectively. Our base editing system in B. subtilis has a 5 nt editing window, which is similar to previous reported base editing in other microorganisms. We demonstrated that the plasmid curing rate is almost 100%, which is advantageous for multiple rounds of genome engineering in B. subtilis. Finally, we applied multiplex genome editing to generate a B. subtilis 168 mutant strain with eight inactive extracellular proteases genes in just two rounds of base editing and plasmid curing, suggesting that it is a powerful tool for gene manipulation in B. subtilis and industrial applications in the future.

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.

CRISPR-Cas systems have become widely used across all fields of biology as a genome engineering tool. With its recent demonstration in the Gram positive industrial workhorse Bacillus subtilis, this tool has become an attractive option for rapid, markerless strain engineering of industrial production hosts. Previously described strategies for CRISPR-Cas9 genome editing in B. subtilis have involved chromosomal integrations of Cas9 and single guide RNA expression cassettes, or construction of large plasmids for simultaneous transformation of both single guide RNA and donor DNA. Here we use a flexible, co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%. This allowed multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homolog, M-protease. A novel subtilisin E variant was obtained with increased thermotolerance and activity.