Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

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Tran, MTN; Khalid, MKNM; Wang, Q; Walker, JKR; Lidgerwood, GE; Dilworth, KL; Lisowski, L; Pebay, A; Hewitt, AWDate
2020-09-25Source Title
Nature CommunicationsPublisher
NATURE RESEARCHAffiliation
Surgery (RMH)Anatomy and Neuroscience
Centre for Eye Research Australia (CERA)
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Tran, M. T. N., Khalid, M. K. N. M., Wang, Q., Walker, J. K. R., Lidgerwood, G. E., Dilworth, K. L., Lisowski, L., Pebay, A. & Hewitt, A. W. (2020). Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing. NATURE COMMUNICATIONS, 11 (1), https://doi.org/10.1038/s41467-020-18715-y.Access Status
Open AccessAbstract
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.
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