Characterization the gene editing with miniature nucleases TnpB, IscB and enIscB in Escherichia coli strains

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 strains. First, the toxicity and cleavage results 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.