Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing
Minh Thuan Nguyen Tran, Mohd Khairul Nizam Mohd Khalid, Qi Wang, Jacqueline K. R. Walker, Grace E. Lidgerwood, Kimberley L. Dilworth, Leszek Lisowski, Alice Pébay, Alex W. Hewitt
Abstract
Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an 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 one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.