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

The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity. Our model involves multiple layers of previously underappreciated cooperativity in BE steps including: (1) Cas9 binding can potentially expose both DNA strands for capture by the deaminase, a feature that is enhanced by guide RNA mismatches; (2) after strand capture, the intrinsic activity of the DNA deaminase can tune window size and base editing efficiency; (3) Cas9 defines the boundaries of editing on each strand, with deamination blocked by Cas9 binding to either the PAM or the protospacer; and (4) non-canonical edits on the guide RNA bound strand can be further elicited by changing which strand is nicked by Cas9. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C>T and G>A transitions over >65 bp with significant potential for targeted gene diversification.