Highly efficient genome editing in Bacillus subtilis via miniature DNA nucleases IscB

Existing CRISPR-based genome editing tools are limited in Bacillus subtilis due to the large cas gene. The recently reported DNA nuclease IscB has the potential to be developed into a novel genome editing tool due to its size being one-third of Cas9, while its application in B. subtilis remains unexplored. In this study, genome editing tools pBsuIscB/pBsuenIscB based on IscB and enIscB (enhanced IscB) were established in B. subtilis SCK6, and successfully deleted 0.6 kb to 4.3 kb genes with efficiencies up to 100%. Subsequently, the pBsuenIscB with higher deletion efficiency was used, whereby the large genomic fragment of 37.7 kb or 169.9 kb was deleted with only one ωRNA. Additionally, single-copy or multi-copy mCherry genes was integrated by using pBsuenIscB. Finally, the editing plasmid was eliminated and the second round of genome editing was completed. Overall, this study has successfully applied IscB to B.subtilis , expanded the genome editing toolbox of B. subtilis , and will help to construct B. subtilis chassis for production of a variety of biomolecules.