The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, pathways have been discovered for DNA repair both with and without a repair template oligonucleotide. We have previously described the ExACT pathway for oligo-driven DNA repair, which consisted of a two-step DNA synthesis-driven repair catalyzed by the simultaneous binding of the repair oligonucleotide (ssODN) upstream and downstream of the double-strand break. In order to better elucidate the mechanism of ExACT-based repair, we have challenged the assumptions of the pathway with those outlines in other similar non-ssODN-based DNA repair mechanisms. This more comprehensive iteration of the ExACT pathway better described the many different ways where DNA repair can occur in the presence of a repair oligonucleotide after CRISPR cleavage, as well as how these previously distinct pathways can overlap and lead to even more unique repair outcomes.
Keywords: CRISPR/Cas; DNA repair; gene repair; homology directed repair; synthesis-dependent strand annealing.