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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.