Structural Insights into DdCBE in Action Enable High-Precision Mitochondrial DNA Editing

DdCBE couples TALE arrays and the 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-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 near-background levels, thereby generating accurate DdCBE (aDdCBE). Using aDdCBE, we precisely introduced a 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.