Structural insights into DdCBE in action enable highprecision mitochondrial DNA editing by Jiangchao Xiang, Wenchao Xu, Jing Wu, Yaxin Luo, Chengyu Liu, Yaofeng Hou, Jia Chen, Bei Yang ISBN 10.1016/J.MOLCEL.2025.08.016 instant download

Structural insights into DdCBE in action enable highprecision mitochondrial DNA editing by Jiangchao Xiang, Wenchao Xu, Jing Wu, Yaxin Luo, Chengyu Liu, Yaofeng Hou, Jia Chen, Bei Yang ISBN 10.1016/J.MOLCEL.2025.08.016 instant download

DddA-derived cytosine base editor (DdCBE) couples transcription activator-like effector (TALE) arrays andthe double-stranded DNA (dsDNA)-specific cytidine deaminase DddA to target mitochondrial DNA (mtDNA)for editing. However, structures of DdCBE in action are unavailable, impeding its mechanistic-based optimization for high-precision-demanding therapeutic applications. Here, we determined the cryo-electron microscopy (cryo-EM) structures of DdCBE targeting two native mitochondrial gene loci and combined editing data from systematically designed spacers to develop WinPred, a model that can predict DdCBE’s editing outcome and guide its design to achieve high-precision editing. Furthermore, structure-guided engineering of DddA narrowed the editing window of DdCBE to 2–3 nt while minimizing its off-target (OT) editing to nearbackground levels, thereby generating accurate DdCBE (aDdCBE). Using aDdCBE, we precisely introduced a Leber hereditary optic neuropathy (LHON)-disease-related mutation into mtDNA and faithfully recapitulated the pathogenic conditions without interference from unintended bystander or OT mutations. Our work provides a mechanistic understanding of DdCBE and establishes WinPred and aDdCBE as useful tools for faithfully modelling or correcting disease-related mtDNA mutations.