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

In this work, Chase Beisel’s lab demonstrates the novel gene editing approach of “append editing”. In contrast to base editing, which changes the DNA base identity directly, append editing uses chemical modification to elicit a desired gene editing outcome. DarT2, a phage ADP-ribosyltransferase was fused to a nickase Cas9 and this fusion could then be targeted to specific DNA sequences to add an ADP-ribosyl molecule to a thymine base on the non-target DNA strand. myTXTL was used to express active DarT2 and then an ADP-ribosylation assay of a ssDNA oligo was performed using the myTXTL reaction products without purification. The ribosylated ssDNA base was then shown to block Klenow DNA polymerase extension on the ssDNA oligo template.