Directing nucleobase deamination or removal can generate precise genomic edits without double-stranded DNA breaks. However, a much broader spectrum of DNA chemical modifications is available that could further expand gene-editing capabilities. In this talk, I will describe my group's effort to evaluate the impact of appending rather than removing a chemical moiety using the bacterial DNA ADP-ribosyl transferase DarT2. DarT2 naturally exists within an anti-phage toxin-antitoxin system that appends a bulky ADP-ribosyl moiety to thymine in single-stranded DNA, interfering with phage replication. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymine. Remarkably, this combination drives editing outcomes that depend on the domain of life and are distinct from base deamination and removal. In tested bacteria, site-specific ADP-ribosylation drives efficient homologous recombination with a provided repair template, offering flexible and scar-free genome editing without base replacement or counterselection. In tested eukaryotes, including yeast, plants and human cells, site-specific ADP-ribosylation preferentially drives replacement of the modified thymine with a bias towards adenine or cytosine, with limited insertions or deletions. Altogether, our approach expands current modalities for precision gene editing by appending chemical moieties to DNA, creating distinct editing opportunities in bacteria and eukaryotes.

Learning Objectives:

• Specify why DarT2 was chosen to explore append editing

• Describe the outcome of targeted DNA ADP-ribosylation in bacteria and eukaryotes

• Compare and contrast append editing to base editing