Publications
bioRxiv : the preprint server for biologyNov 2024 DOI:
10.1101/2024.11.17.623984

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

Patinios, Constantinos; Gupta, Darshana; Bassett, Harris V; Collins, Scott P; Kamm, Charlotte; Kibe, Anuja; Wang, Yanyan; Zhao, Chengsong; Vollen, Katie; Toussaint, Christophe; Polkoff, Kathryn M; Nguyen, Thuan; Calvin, Irene; Migur, Angela; Al'Abri, Ibrahim S; Achmedov, Tatjana; Del Re, Alessandro; Saliba, Antoine-Emmanuel; Crook, Nathan; Stepanova, Anna N; Alonso, Jose M; Beisel, Chase L
Product Used
Genes
Abstract
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 anti-phage 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 efficient homologous recombination in tested bacteria, offering flexible and scar-free genome editing without base replacement nor counterselection. In tested eukaryotes including yeast, plants 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 a chemical moiety to DNA to expand current modalities for precision gene editing.
Product Used
Genes

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