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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

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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 Hewitt
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.

History

Publication title

Nature Communications

Volume

11

Article number

4871

Number

4871

Pagination

1-10

ISSN

2041-1723

Department/School

Menzies Institute for Medical Research

Publisher

Nature Publishing Group

Place of publication

United Kingdom

Rights 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

Socio-economic Objectives

Clinical health not elsewhere classified

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