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<p>DNA endonucleases TnpB and IscB are emerging candidates for combating drug-resistant bacteria, particularly <i>Escherichia coli</i>, due to their specificity in targeting DNA and smaller size. However, the genome-editing of TnpB/IscB in <i>E. coli</i> remains unclear. This study characterized the genome editing of TnpB/IscB in different <i>E. coli</i> 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 <i>E. coli</i> during TnpB/IscB editing was elucidated. Overall, this study successfully applied TnpB/IscB/enIscB to genome editing in <i>E. coli</i>, which will expand the genetic manipulation toolbox in <i>E. coli</i> and facilitate the development of the antimicrobial drugs.</p>

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Characterization of the genome editing with miniature DNA nucleases TnpB and IscB in Escherichia coli strains

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年2月19日

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  • Hongjie Tang,
  • Jie Gao,
  • Hengyi Wang,
  • Mingjun Sun,
  • Suyi Zhang,
  • Chuan Song,
  • Qi Li

摘要

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.