Engineering a miniaturized SaCas9 adenine base editor with reduced RNA off-targets and increased on-target DNA editing efficiencies

We compared the editing profile of circularly permuted and domain inlaid Cas9 base editors, and found that on-target editing can be largely maintained following their intradomain insertion, but that structural permutation of the adenine base editor (ABE) can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer a SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents the smallest Cas9-ABE available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry