141152 - Engineering domain-inlaid SaCas9 adenine.pdf (1.09 MB)
Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing
journal contribution
posted on 2023-05-20, 18:16 authored by Minh Thuan Nguyen Tran, Mohd Khalid, MKN, Wang, Qi, Walker, JKR, Lidgerwood, GE, Dilworth, KL, Lisowski, L, Pebay, A, Alexander HewittAlexander HewittPrecision 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.
History
Publication title
Nature CommunicationsVolume
11Article number
4871Number
4871Pagination
1-10ISSN
2041-1723Department/School
Menzies Institute for Medical ResearchPublisher
Nature Publishing GroupPlace of publication
United KingdomRights statement
© The Author(s) 2020. Licensed under Creative Commons Attribution 4.0 International (CC BY 4.0) http://creativecommons.org/licenses/by/4.0/Repository Status
- Open