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<p>Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here we harness the bacterial antiphage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives homologous recombination, offering flexible and scar-free genome editing without base replacement or counterselection. In tested yeast, plant and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of chemical moieties to DNA to expand current modalities for precision gene editing.</p>

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Targeted DNA ADP-ribosylation triggers templated repair in bacteria and base mutagenesis in eukaryotes

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年9月4日

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  • Darshana Gupta,
  • Constantinos Patinios,
  • Harris V. Bassett,
  • Anuja Kibe,
  • Scott P. Collins,
  • Charlotte Kamm,
  • Yanyan Wang,
  • Chengsong Zhao,
  • Katie Vollen,
  • Christophe Toussaint,
  • Irene Calvin,
  • Grégoire Cullot,
  • Eric J. Aird,
  • Kathryn M. Polkoff,
  • Thuan Phu Nguyen-Vo,
  • Angela Migur,
  • Friso Schut,
  • Ibrahim S. Al’Abri,
  • Tatjana Achmedov,
  • Alessandro Del Re,
  • Jacob E. Corn,
  • Antoine-Emmanuel Saliba,
  • Nathan Crook,
  • Anna N. Stepanova,
  • Jose M. Alonso,
  • Chase L. Beisel

摘要

Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here we harness the bacterial antiphage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives homologous recombination, offering flexible and scar-free genome editing without base replacement or counterselection. In tested yeast, plant and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of chemical moieties to DNA to expand current modalities for precision gene editing.