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

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 developme...

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Detalles Bibliográficos
Autores principales: Willis, Julian C. W., Silva-Pinheiro, Pedro, Widdup, Lily, Minczuk, Michal, Liu, David R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9684478/
https://www.ncbi.nlm.nih.gov/pubmed/36418298
http://dx.doi.org/10.1038/s41467-022-34784-7
Descripción
Sumario: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.