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

Julian C. W. Willis, Pedro Silva-Pinheiro, Lily Widdup, Michal Minczuk and David R. Liu ()
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Julian C. W. Willis: Broad Institute of MIT and Harvard
Pedro Silva-Pinheiro: University of Cambridge
Lily Widdup: Broad Institute of MIT and Harvard
Michal Minczuk: University of Cambridge
David R. Liu: Broad Institute of MIT and Harvard

Nature Communications, 2022, vol. 13, issue 1, 1-16

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-34784-7

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