Abstract
DNA endonucleases TnpB and IscB are emerging candidates for combating drug-resistant bacteria, particularly Escherichia coli, due to their specificity in targeting DNA and smaller size. However, the genome-editing of TnpB/IscB in E. coli remains unclear. This study characterized the genome editing of TnpB/IscB in different E. coli strains. First, the toxicity and cleavage results indicated TnpB was effective only in MG1655, whereas IscB and enIscB demonstrated functionality in ATCC9637/BL21(DE3). Subsequently, a genome-editing tool was established in MG1655 by using TnpB (as a thermophilic programmable endonuclease), achieving up to 100% editing efficiency, while IscB/enIscB achieved editing in ATCC9637/BL21(DE3). Additionally, the editing plasmids were successfully cured. Finally, the mechanism underlying the escape of E. coli during TnpB/IscB editing was elucidated. Overall, this study successfully applied TnpB/IscB/enIscB to genome editing in E. coli, which will expand the genetic manipulation toolbox in E. coli and facilitate the development of the antimicrobial drugs.