Targeted DNA ADP ribosylation drives distinct editing outcomes in bacteria and eukaryotes

By fusing an attenuated DarT2 to a nicking Cas9, we program site-specific ADP-ribosylation that drives editing outcomes distinct from base deamination or removal and unexpectedly depend on the domain of life. In tested bacteria, site-specific ADP-ribosylation drives efficient homologous recombination with a provided recombineering template, offering flexible and scar-free genome editing without base replacement or counterselection. In tested eukaryotes including yeast, plants and human cell lines, site-specific ADP-ribosylation preferentially drives replacement of the ADP-ribosylated thymine with a bias toward adenine or cytosine, with minimal insertions or deletions. Our approach thus expands current modalities for precision gene editing by appending chemical groups to DNA, creating distinct editing opportunities in bacteria and eukaryotes.