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

Abstract

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.