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

• Published in: Nature communications 2022-11, Vol.13 (1), p.7204-16, Article 7204
• Main Authors: Willis, Julian C. W., Silva-Pinheiro, Pedro, Widdup, Lily, Minczuk, Michal, Liu, David R.
• Format: Article
• Language: English
• Subjects: 45; 45/23; 45/41; 631/1647/1511; 631/61/201/2110; Animals; Arrays; Binding; CRISPR; CRISPR-Cas Systems - genetics; Cytidine deaminase; Cytosine; Cytosine - metabolism; Deoxyribonucleic acid; DNA; DNA - genetics; DNA - metabolism; DNA damage; DNA-binding protein; Editing; Gene Editing; Humanities and Social Sciences; Mice; Mitochondria; Mitochondria - genetics; Mitochondria - metabolism; Mitochondrial DNA; multidisciplinary; Muscles; Mutation; Proteins; Science; Science (multidisciplinary); Skeletal muscle; Therapeutic applications; Uracil; Zinc; Zinc finger proteins; Zinc Fingers - genetics
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-34784-7

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. Zinc finger (ZF) arrays are programmable DNA-binding proteins. Here the authors report ZF-DddA-derived cytosine base editors (DdCBEs) and optimise their architectures to improve targeting; they apply these variants in vitro and in vivo to mitochondrial base editing and show higher editing than ZF deaminases.