Data availabilityRaw data of whole mitochondrial genome sequencing for off-target analysis are available as a BioProject with the project identifier PRJCA038551 (https://ngdc.cncb.ac.cn/gsa-human/s/88Zs4Xcl) in the China National Center for Bioinformation–National Genomics Data Center database55,56. Cryo-EM density maps and structure coordinates have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank, with accession codes EMD-62996 and PDB 9LCY for TOD6inact with the TC dsDNA substrate; EMD-62999 and PDB 9LD1 for TOD4inact with the TC dsDNA substrate, along with a local map of the orienting domain and the deaminase region; EMD-62997 and PDB 9LCZ for TOD6inact with the GC dsDNA substrate; EMD-62995 and PDB 9LCX for TOD6inact with the AC dsDNA substrate; and EMD-62998 and PDB 9LD0 for TOD6inact with the CC dsDNA substrate, and corresponding cryo-EM maps, half-maps and models are also provided through Figshare (https://figshare.com/s/2f675005689b82275513)57. 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This work was funded by the National Key Research and Development Program of China (2024YFC3405500 to L.M. and 2021YFA1100200 to Y.W.), the National Natural Science Foundation of China (32525039, 32430063 and 22137005 to P.L.; 32025007 and 32130017 to Y.W.; 82271897 to M.J.), the State Key Laboratory of Gene Expression (grant no. 2025ZY01117), the Zhejiang Provincial Natural Science Foundation of China (LR23C050001 to P.L. and LZYQ25C070001 to M.J.), the ‘Pioneer’ and ‘Leading Goose’ R&D Program of Zhejiang (grant no. 2024SSYS0036), the China Postdoctoral Science Foundation (2024M762947 to L.M.) and the Westlake Center for Genome Editing of Westlake University, program no. 21200000A992410/003. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.Author informationAuthor notesThese authors contributed equally: Li Mi, Yu-Xuan Li, Xinchen Lv.Authors and AffiliationsState Key Laboratory of Gene Expression, Research Center for Industries of the Future, School of Life Sciences, Westlake University, Hangzhou, ChinaLi Mi, Xinchen Lv, Xu Liu, Kairan Zhang, Huican Li, Yue Yao, Leping Zhang, Zhe Xu, Min Jiang & Peilong LuWestlake Laboratory of Life Sciences and Biomedicine, Hangzhou, ChinaLi Mi, Xinchen Lv, Xu Liu, Kairan Zhang, Huican Li, Yue Yao, Leping Zhang, Zhe Xu, Min Jiang & Peilong LuInstitute of Biology, Westlake Institute for Advanced Study, Hangzhou, ChinaLi Mi, Xinchen Lv, Xu Liu, Kairan Zhang, Huican Li, Yue Yao, Leping Zhang, Zhe Xu, Min Jiang & Peilong LuState Key Laboratory of Gene Function and Modulation Research, Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, ChinaYu-Xuan Li, Zi-Li Wan & Yangming WangBeijing Advanced Center of RNA Biology (BEACON), Peking University, Beijing, ChinaYu-Xuan Li, Zi-Li Wan & Yangming WangDepartment of Neurology, Shandong Key Laboratory of Mitochondrial Medicine and Rare Diseases, Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital of Shandong University, Jinan, ChinaXingyu Zhuang & Kunqian JiShandong Key Laboratory, Magnetic Field-free Medicine and Functional Imaging, Qilu Hospital of Shandong University, Jinan, ChinaXingyu Zhuang & Kunqian JiSouthwest United Graduate School, Kunming, ChinaYangming WangAuthorsLi MiView author publicationsSearch author on:PubMed Google ScholarYu-Xuan LiView author publicationsSearch author on:PubMed Google ScholarXinchen LvView author publicationsSearch author on:PubMed Google ScholarZi-Li WanView author publicationsSearch author on:PubMed Google ScholarXu LiuView author publicationsSearch author on:PubMed Google ScholarKairan ZhangView author publicationsSearch author on:PubMed Google ScholarHuican LiView author publicationsSearch author on:PubMed Google ScholarYue YaoView author publicationsSearch author on:PubMed Google ScholarLeping ZhangView author publicationsSearch author on:PubMed Google ScholarZhe XuView author publicationsSearch author on:PubMed Google ScholarXingyu ZhuangView author publicationsSearch author on:PubMed Google ScholarKunqian JiView author publicationsSearch author on:PubMed Google ScholarMin JiangView author publicationsSearch author on:PubMed Google ScholarYangming WangView author publicationsSearch author on:PubMed Google ScholarPeilong LuView author publicationsSearch author on:PubMed Google ScholarContributionsL.M., Y.W. and P.L. conceived the research and designed experiments. L.M. designed the orienting domains. L.M., Y.-X.L. and Z.-L.W. completed molecular cloning. L.M. performed the E. coli-based editing assay and protein purification. X. Lv performed the in vitro deamination assay. H.L. prepared cryo-EM samples. X. Lv and L.M. collected cryo-EM data and solved the EM structures. Y.-X.L. performed the mammalian cell base-editing assay with assistance from Z.-L.W. Y.-X.L. constructed off-target editing libraries. L.M. and Y.-X.L. analyzed the sequencing data. Y.Y. provided assistance in experiments. X. Liu, L.Z., Z.X. and M.J. characterized the application of DdCBE–TODs in MEF cells and mice. X.Z. and K.J. provided primary skin fibroblasts with the m.A8344G mutation. K.Z. carried out molecular dynamics simulations. P.L., Y.W. and L.M. wrote the manuscript with X. Lv’s and Y.-X.L.’s assistance.Corresponding authorsCorrespondence to Yangming Wang or Peilong Lu.Ethics declarationsCompeting interestsL.M. and P.L. are inventors on a provisional patent application submitted by Westlake University; the remaining authors declare no competing interests.Peer reviewPeer review informationNature Structural & Molecular Biology thanks Sangsu Bae, Kozo Tomita, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Sara Osman and Melina Casadio, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 mDdCBEs using a truncated Ddd_Ss variant demonstrate significant mtDNA editing activity.a, Architectures of DdCBE and mDdCBEs for mtDNA editing. A truncated Ddd_Ss variant (Ddd_Ss_S, denoting Ddd_Ss_Short, residues 2–114), which lacks the C-terminal SPKK-related motif, was utilized to construct mDdCBEs. MTS, mitochondrial localization sequences. b, Heat maps showing C•G-to-T•A conversion frequencies for DdCBE and mDdCBEs at the MT-ND6 site in mtDNA of HEK293T cells. Shown are individual values from n = 3 independent experiments. c, Indel frequencies of DdCBE and mDdCBEs at the MT-ND6 site in mtDNA. Shown are mean ± SD from n = 3 independent experiments. The transfection duration was 3 days. Source data are provided as a Source Data file.Source dataExtended Data Fig. 2 Base editing by mDdCBE-TODs in E. coli-based editing assays.a, b, The editing efficiency of mDdCBE and five designer mDdCBE-TODs at various positions of the four DNA substrates (as shown in Fig. 1c). The numbers on the X-axis represent the distance, measured in nucleotides, to the last nucleotide in the TALE binding sequence. mDdCBE and mDdCBE-TODs were induced with 0.02 g/L (a) or 0.2 g/L (b) arabinose for 1 h. Data are presented as mean ± SD from n = 3 independent experiments. Source data are provided as a Source Data file.Source dataExtended Data Fig. 3 In vitro cytosine deamination assays for mDdCBE and TOD6.a, In vitro cytosine deamination activity of mDdCBE and TOD6. dsDNA substrates (S1–S4) were labeled with 6-Carboxyfluorescein (FAM, shown as a green star). The DNA sequence is presented at the top, with cytosines highlighted in red. The white bar denotes the TALE binding sequence, and the purple bar denotes the complementary sequence. Cytosine deamination results in cleavage by further treatment (Methods), generating cleaved products (P) with increased mobility. b, In vitro cytosine deamination activity of TOD6 against four dsDNA substrates (S) with 5′-GC, AC, TC, or CC target sequences. Representative gel images from n = 3 independent experiments are shown. Source data are provided as a Source Data file.Source dataExtended Data Fig. 4 Cryo-EM structure of TOD4 complexed with dsDNA substrate.a, Cryo-EM structure of TOD4inact complexed with dsDNA substrate (colored) is in close agreement with the design model (gray). Schematic diagram (top) shows the domain organization of TOD4, comprising TALE, orienting domain C4 (candidate 4), and Ddd_Ss_S deaminase. b, The zoomed view showing the last TALE repeat (purple), orienting domain C4 (blue) and inactive Ddd_Ss_S (pink) in TOD4 interacting with dsDNA substrate (orange). c, d, Detailed views showing that residues from the orienting domain C4 form extensive interactions with both the last TALE repeat and the Ddd_Ss_S deaminase. e, Cryo-EM structures of Ddd_Ss_S in TOD6inact and TOD4inact complexed with dsDNA substrate are almost identical.Extended Data Fig. 5 mtDNA editing by mDdCBE-TOD6 in HEK293T cells.a, Architectures of mDdCBE and mDdCBE-TOD6 for mtDNA editing. b–f, mtDNA editing by mDdCBE (pink) and mDdCBE-TOD6 (blue) at the MT-CYB (b), MT-ND1.1 (c), MT-ND3 (d), MT-ND4 (e) and MT-TK.1 (f) sites. In panel (c), a putative TALE binding site that is highly similar to the designated TALE binding site is indicated. Sequencing data from untreated cells (grey) are shown as control. Data are presented as mean ± SD from n = 3 independent experiments. The transfection duration was 3 days. Source data are provided as a Source Data file.Source dataExtended Data Fig. 6 On-target and off-target editing activities of mDdCBE-TOD6.a–f, Average C•G to T•A editing efficiencies at on-target (red dots, designated target site) and off-target (gray dots) sites across mtDNA for MT-CYB-mDdCBE (a), MT-CYB-mDdCBE-TOD6 (b), MT-ND1.1-mDdCBE (c), MT-ND1.1-mDdCBE-TOD6 (d), MT-ND3-mDdCBE (e) and MT-ND3-mDdCBE-TOD6 (f). Sites with average editing frequency greater than 1% are shown. Data are shown as means from n = 2 independent experiments. g, Venn diagram showing the overlap of off-target sites among MT-CYB-mDdCBE, MT-ND1.1-mDdCBE and MT-ND3-mDdCBE. h, Venn diagram showing the overlap of off-target sites among MT-CYB-mDdCBE-TOD6, MT-ND1.1-mDdCBE-TOD6 and MT-ND3-mDdCBE-TOD6. Source data are provided as a Source Data file.Source dataExtended Data Fig. 7 Splitting at N67 (DdCBE-TOD6_N67) achieves efficient and precise editing.a, Architectures of split DdCBE_S, DdCBE, DdCBE-TOD6_S, and DdCBE-TOD6 at the C-terminal of N29 or N94 for E. coli-based editing assays. b, The editing efficiency of N29 and N94 splits in E. coli. Base editors were induced with 0.2 g/L arabinose for 1 h. c, The editing efficiency of N-half and C-half components of the N29 and N94 splits in E. coli. DdCBE and DdCBE-TOD6 share the same C-half split. Base editors were induced with 0.2 g/L arabinose for 1 h. d, Schematic representation of DdCBE_N29 and DdCBE-TOD6_N29 for mtDNA editing. e, f, The mtDNA editing efficiencies of DdCBE_N29 and DdCBE-TOD6_N29 at the MT-CYB (e) and MT-TS2 (f) site in HEK293T cells. The transfection duration was 3 days. g, h, The editing efficiency of DdCBE-TOD6_N67 split and its N-half and C-half components in E. coli. DdCBE-TOD6 variants were induced with 0.2 g/L arabinose for 1 h (g) or 3 h (h). Data are presented as mean ± SD from n = 3 independent experiments. Source data are provided as a Source Data file.Source dataExtended Data Fig. 8 Editing activities of DdCBE-TOD6_N67-derived constructs for mtDNA.a, Architectures of DdCBE and DdCBE-TOD6_N67-derived constructs. b–d, mtDNA editing efficiencies of DdCBE and DdCBE-TOD6_N67-derived constructs at the MT-CYB and MT-TS2 sites in HEK293T cells. Data are presented as mean ± SD from n = 3 independent experiments. Source data are provided as a Source Data file.Source dataExtended Data Fig. 9 DdCBE-TOD6-derived constructs achieve single-nucleotide precision editing at non-CC context positions.a–e, mtDNA editing efficiencies of DdCBE_N94 and DdCBE-TOD6-derived constructs at the MT-CO3 (a), MT-ND5 (b), MT-ND1.1 (c), MT-ND1.2 (d) and MT-ND1.3 (e) sites in HEK293T cells. Note that MT-ND5 site contains a CCCC sequence, and MT-ND1.3 site contains an ACC sequence. f–h, mtDNA editing efficiencies of DdCBE_N94 and DdCBE-TOD6-derived constructs at the mt-Nd1.1 (f), mt-Nd1.2 (g) and mt-Nd6 (h) sites in NIH/3T3 cells. Data are presented as mean ± SD from n = 3 independent experiments. The transfection duration was 3 days. i, Distribution of disease-associated mtDNA mutations across different sequence contexts. Source data are provided as a Source Data file.Source dataExtended Data Fig. 10 DdCBE-TODs achieve high precision mtDNA editing in human primary skin fibroblast and MEF cells.a, Average mtDNA genome-wide C•G to T•A conversion rate induced by DdCBE-TOD6_sCUTV and DdCBE-TOD6_dNU for MT-TK.2 sites in human primary skin fibroblasts. Naturally occurring single nucleotide variations (SNVs) with heteroplasmy fraction >10% were excluded from the analysis. Data represent the mean from n = 2 independent experiments. b, m.G8369 mutation load in untreated and DdCBE-TOD6_sCUT-treated MEF clones. Each dot represents the mutation load measured from a single-cell-derived clone. Successfully edited MEF clones by DdCBE-TOD6_sCUT exhibited mutation loads ranging from 42.01% to 89.89%, with a median mutation load of 70.05%. Median with interquartile range is shown, n = 4 for untreated, n = 53 for DdCBE-TOD6_sCUT. c, Heat map showing C•G-to-T•A conversion frequencies for 4 untreated clones and 4 clones with the highest mutation loads after DdCBE-TOD6_sCUT treatment, as depicted in panel (b). Shown are individual values. Source data are provided as a Source Data file.Source dataSupplementary informationSupplementary InformationSupplementary Figs. 1–5, Sequences 1–3 and NoteReporting SummaryPeer Review FileSupplementary Table 1MitoTALE binding sites.Supplementary Table 2Primers used in this paper.Source dataSource Data Fig. 4Statistical source data.Source Data Fig. 5Statistical source data.Source Data Fig. 6Statistical source data.Source Data Extended Data Fig. 1Statistical source data.Source Data Extended Data Fig. 2Statistical source data.Source Data Extended Data Fig. 3Unprocessed gels.Source Data Extended Data Fig. 5Statistical source data.Source Data Extended Data Fig. 6Statistical source data.Source Data Extended Data Fig. 7Statistical source data.Source Data Extended Data Fig. 8Statistical source data.Source Data Extended Data Fig. 9Statistical source data.Source Data Extended Data Fig. 10Statistical source data.Rights and permissionsSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Reprints and permissionsAbout this article