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Compact zinc finger base editors that edit mitochondrial or nuclear DNA in vitro and in vivo.

Published version
Peer-reviewed

Type

Article

Change log

Authors

Silva-Pinheiro, Pedro  ORCID logo  https://orcid.org/0000-0002-0872-5749
Widdup, Lily 

Abstract

DddA-derived cytosine base editors (DdCBEs) use programmable DNA-binding TALE repeat arrays, rather than CRISPR proteins, a split double-stranded DNA cytidine deaminase (DddA), and a uracil glycosylase inhibitor to mediate C•G-to-T•A editing in nuclear and organelle DNA. Here we report the development of zinc finger DdCBEs (ZF-DdCBEs) and the improvement of their editing performance through engineering their architectures, defining improved ZF scaffolds, and installing DddA activity-enhancing mutations. We engineer variants with improved DNA specificity by integrating four strategies to reduce off-target editing. We use optimized ZF-DdCBEs to install or correct disease-associated mutations in mitochondria and in the nucleus. Leveraging their small size, we use a single AAV9 to deliver into heart, liver, and skeletal muscle in post-natal mice ZF-DdCBEs that efficiently install disease-associated mutations. While off-target editing of ZF-DdCBEs is likely too high for therapeutic applications, these findings demonstrate a compact, all-protein base editing research tool for precise editing of organelle or nuclear DNA without double-strand DNA breaks.

Description

Keywords

Mice, Animals, Gene Editing, CRISPR-Cas Systems, DNA, Zinc Fingers, Cytosine, Mitochondria

Journal Title

Nat Commun

Conference Name

Journal ISSN

2041-1723
2041-1723

Volume Title

13

Publisher

Springer Science and Business Media LLC
Sponsorship
Medical Research Council (MC_UU_00015/4)
Medical Research Council (MC_UU_00015/7)
MRC (MC_UU_00028/3)