9JO8

Cryo-EM structure of the mono-DdCBE bound to a dsDNA substrate.

This is a non-PDB format compatible entry.

Summary for 9JO8

• Entry DOI: 10.2210/pdb9jo8/pdb
• EMDB information: 61646
• Descriptor: TALE repeat protein targeting mitochondiral ND1-L gene., TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND4 gene sequence., a complementary strand of TALE repeat protein recognized single-strand DNA sequence and a complementary strand of mitochondrial ND4 gene sequence., ... (6 entities in total)
• Functional Keywords: tale, ddda deaminase, ddcbe, dsdna, nd4, mitochondrial base editor., dna binding protein/dna, dna binding protein-dna complex
• Biological source: Xanthomonas; More
• Total number of polymer chains: 4
• Total formula weight: 104853.22

Authors

Jiangchao, X.,Wenchao, X.,Jia, C.,Bei, Y. (deposition date: 2024-09-24, release date: 2025-09-17, Last modification date: 2025-10-08)

Primary citation

Xiang, J.,Xu, W.,Wu, J.,Luo, Y.,Liu, C.,Hou, Y.,Chen, J.,Yang, B.
Structural insights into DdCBE in action enable high-precision mitochondrial DNA editing.
Mol.Cell, 85:3357-, 2025

PubMed Abstract: DddA-derived cytosine base editor (DdCBE) couples transcription activator-like effector (TALE) arrays and the 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 near-background 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 modeling or correcting disease-related mtDNA mutations.

PubMed: 40934924
DOI: 10.1016/j.molcel.2025.08.016

Experimental method

ELECTRON MICROSCOPY (3 Å)