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

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Nguyen Tran, MT, Mohd Khalid, MKN, Wang, Q ORCID: 0000-0003-0397-6554, Walker, JKR, Lidgerwood, GE, Dilworth, KL, Lisowski, L, Pebay, A and Hewitt, AW ORCID: 0000-0002-5123-5999 2020 , 'Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing' , Nature Communications, vol. 11, no. 1 , pp. 1-10 , doi: 10.1038/s41467-020-18715-y.

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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.

Item Type: Article
Authors/Creators:Nguyen Tran, MT and Mohd Khalid, MKN and Wang, Q and Walker, JKR and Lidgerwood, GE and Dilworth, KL and Lisowski, L and Pebay, A and Hewitt, AW
Journal or Publication Title: Nature Communications
Publisher: Nature Publishing Group
ISSN: 2041-1723
DOI / ID Number: 10.1038/s41467-020-18715-y
Copyright Information:

© The Author(s) 2020. Licensed under Creative Commons Attribution 4.0 International (CC BY 4.0) http://creativecommons.org/licenses/by/4.0/

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