Targeted DNA ADP-ribosylation triggers templated repair in bacteria and base mutagenesis in eukaryotes
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.
Authors
- Tatjana Achmedov
- Eric J. Aird (ORCID: https://orcid.org/0000-0002-4873-042X)
- José M. Alonso (ORCID: https://orcid.org/0000-0001-7087-1571)
- Grégoire Cullot (ORCID: https://orcid.org/0000-0003-3925-705X)
- Ibrahim S. Al’Abri (ORCID: https://orcid.org/0000-0003-2600-9612)
- Scott P. Collins (ORCID: https://orcid.org/0000-0001-9359-5530)
- Thuan Phu Nguyen-Vo (ORCID: https://orcid.org/0000-0002-4165-6896)
- Christophe Toussaint (ORCID: https://orcid.org/0000-0003-3419-490X)
- Friso T Schut
- Kathryn M. Polkoff (ORCID: https://orcid.org/0000-0001-6227-622X)
- Harris V. Bassett
- Anuja Kibe (ORCID: https://orcid.org/0000-0001-8547-785X)
- Chase L. Beisel (ORCID: https://orcid.org/0000-0003-0650-9943)
- Nathan Crook (ORCID: https://orcid.org/0000-0001-6165-1972)
- Jacob E. Corn (ORCID: https://orcid.org/0000-0002-7798-5309)
- Antoine‐Emmanuel Saliba (ORCID: https://orcid.org/0000-0001-8539-2784)
- Anna N. Stepanova (ORCID: https://orcid.org/0000-0003-1018-4758)
- Darshana Gupta
- Constantinos Patinios (ORCID: https://orcid.org/0000-0002-9306-4593)
- Angela Migur
- Katie Vollen
- Yanyan Wang (ORCID: https://orcid.org/0000-0001-8260-5959)
- Chengsong Zhao (ORCID: https://orcid.org/0000-0002-1289-9696)
- Charlotte Kamm
- Alessandro Del Re
- Irene Calvin
Institutions
- North Carolina State University (US)
- University of Würzburg (DE)
- ETH Zurich (CH)
- Helmholtz Centre for Infection Research (DE)
- Helmholtz Institute for RNA-based Infection Research (DE)
Publication Details
- Journal
- Nature Biotechnology
- Published
- 2025-09-04
- DOI
- https://doi.org/10.1038/s41587-025-02802-w
- Citations
- 2
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 1.61
Funders
- National Science Foundation
- North Carolina Biotechnology Center
- European Commission
- Deutscher Akademischer Austauschdienst
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
- NOMIS Stiftung
- Julius-Maximilians-Universität Würzburg
- Vogel Stiftung
- National Institutes of Health
- Elitenetzwerk Bayern