Cooperativity between Cas9 and AID establishes broad and diversifying mutational footprints in base editors

The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now awell-established method to enable targeted genomic base editing. However, anunderstanding of how Cas9 and DNA deaminases collaborate to shape base editor(BE) outcomes has been lacking. Here, we employ a range of hyperactive AID baseeditors (hBEs) to deduce a novel mechanistic model that reveals multiple layers ofpreviously underappreciated cooperativity in BE steps. These include: (1) Cas9 binding can potentially expose both DNA strands for capture by the deaminase, a feature enhanced by guide mismatches; (2) after strand capture, intrinsic deaminase activity can tune window size and base editing activity; and (3) non-canonical base edits can be further elicited by modulating Cas9 nicking activity. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C-to-T and G-to-A transitions over 65 bp with the potential for gene diversification.