Bacillus subtilis genome editing using ssDNA with short homology regions

In this study, we developed a simple and efficient Bacillus subtilis genome editing method in which targeted gene(s) could be inactivated by single-stranded PCR product(s) flanked by short homology regions and in-frame deletion could be achieved by incubating the transformants at 42°C. In this process, homologous recombination (HR) was promoted by the lambda beta protein synthesized under the control of promoter P(RM) in the lambda cI857 P(RM)–P(R) promoter system on a temperature sensitive plasmid pWY121. Promoter P(R) drove the expression of the recombinase gene cre at 42°C for excising the floxed (lox sites flanked) disruption cassette that contained a bleomycin resistance marker and a heat inducible counter-selectable marker (hewl, encoding hen egg white lysozyme). Then, we amplified the single-stranded disruption cassette using the primers that carried 70 nt homology extensions corresponding to the regions flanking the target gene. By transforming the respective PCR products into the B. subtilis that harbored pWY121 and incubating the resultant mutants at 42°C, we knocked out multiple genes in the same genetic background with no marker left. This process is simple and efficient and can be widely applied to large-scale genome analysis of recalcitrant Bacillus species.