Li, Q.; Tang, H.; Gao, J.; Sun, M.

DNA nucleases TnpB and IscB were regarded as new antibacterial strategy to combat the drug-resistant bacteria represented by Escherichia coli due to its specificity in targeting DNA and smallest size, but the genome-editing of TnpB/IscB in E. coli remains unclear. This study characterized the genome-editing of TnpB/IscB in E. coli showed TnpB only worked in E. coli MG1655, while IscB and enIscB could perform in ATCC9637 and BL21(DE3). Next, TnpB-based genome-editing tool was established in MG1655, while IscB/enIscB achieved in ATCC9637/BL21(DE3). The copy number of TnpB/IscB/enIscB were changed to explore the impact of editing efficiency. Moreover, the editing plasmids were successfully cured. Finally, the escaping mechanism of E. coli under editing of TnpB/IscB was revealed. Overall, this study successfully applied TnpB/IscB/enIscB to genome-editing in E. coli, which will broaden genetic manipulation toolbox in E. coli and facilitate the development of new antimicrobial drugs.