Abstract
Transcription activator-like effector-linked deaminases (TALEDs) use their single-stranded DNA (ssDNA)-specific adenosine deaminase TadA8e to mediate A-to-G editing in mitochondrial DNA (mtDNA). The working mechanism of this process is unknown, hindering the development of more effective TALEDs. Here we reveal that TALED-mediated A-to-G editing relies on the formation of an ssDNA region through base excision repair (BER), which is triggered by double-stranded DNA-specific cytidine deaminase (DddA)-induced C-to-U deamination. We develop a series of enhanced TALEDs (eTALED6s) with increased editing efficiency by replacing DddA with the high-activity variant DddA6 and fusing human uracil DNA glycosylase to TadA8e. By further engineering TadA8e, the resulting eTALED6Rs induces efficient on-target editing with reduced bystander editing and off-target editing at the DNA and RNA levels. Lastly, we use eTALED6 and eTALED6R to install a pathogenic mutation in mtDNA. Revealing the mechanism of TALED-mediated A-to-G editing demonstrates that enhancing BER increases editing efficiency.
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Data availability
Targeted deep sequencing data, whole-transcriptome sequencing data and whole-genome sequencing data can be accessed from the the National Center for Biotechnology Information under BioProjects PRJNA1223321, PRJNA1221787 and PRJNA1221509. SNV data from dbSNP (version 151; https://ncbi.nlm.nih.gov/snp/), integrated into the published BEIDOU toolkit, were used to identify de novo SNVs. Source data are provided with this paper.
Code availability
The custom Perl and Shell scripts for calculating frequencies of base substitution are available from GitHub (https://github.com/YangLab/CFBI).
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Acknowledgements
This work is supported by the National Key Research and Development Program of China (2024YFA1803301 to J.C., 2024YFC3405902 to L.Y., 2023YFC3403402 to J.C. and 2023ZD0500501 to B.Y.), National Natural Science Foundation of China (32371514 to J.C., 32430018 to L.Y. and 32070170 to B.Y.), Ministry of Agriculture and Rural Affairs of the People’s Republic of China (NK2022010207 to J.C.), Shanghai Municipal Science and Technology Commission (23JS1400300 and 23DX1900102 to L.Y. and 23ZR1442500 to B.Y.) and Program of Shanghai Academic/Technology Research Leader (23XD1422500 to J.C.). We thank Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine, ShanghaiTech University and Molecular and Cell Biology Core Facility, School of Life Science and Technology, ShanghaiTech University for the technical support. We thank the Biomedical Big Data Platform at the Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University for their deep sequencing services. FACS was provided by the Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, and we thank P. W. Zhang and L. S. Zhang for their assistance with cell sorting.
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Contributions
Y.F. and W.X. conceptualized the project. Y.F. and W.X. designed the experiments. H.Q., X.W., Q.N., L.Z. and J.Wu performed the experiments. J.Wei prepared the libraries for deep sequencing and B.-Q.G. performed the data analysis, supervised by L.Y. J.X. and B.Y. provided technical support. J.C. supervised the work. J.C., Y.F. and W.X. wrote the paper with input from all authors.
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J.C., L.Y. and B.Y. are scientific cofounders of CorrectSequence Therapeutics, a company that uses gene-editing technologies, and members of its scientific advisory board. The other authors declare no competing interests.
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Nature Biotechnology thanks Hyongbum Kim, Bin Shen and Yangming Wang for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 TALED, but not DdCBE, induced bystander editing in TALE-binding regions at other target sites.
a, Schematic of the constructions of sTALED and DdCBE targeting ND4 site. NTD, N-terminal domain of TALE array; CTD, C-terminal domain of TALE array; MTS, mitochondrial targeting sequence; UGI, uracil glycosylase inhibitor. b, A-to-G editing frequencies induced by sTALED and C-to-T base editing frequencies induced by DdCBE at the ND4 site in 293FT cells. NT, non-transfected control. c, Schematic of the constructions of sTALED and DdCBE targeting ND5.3 site. d, A-to-G editing frequencies induced by sTALED and C-to-T base editing frequencies induced by DdCBE at the ND5.3 site in 293FT cells. The sequences of TALE-binding regions are in blue and bystander editing can be found in the boxes with dashed line. The data are presented as the mean ± s.d. from three independent experiments.
Extended Data Fig. 2 Purification of TALED/DdCBE-related proteins.
a, Size-exclusion chromatography profile of the His-tagged L_TALE-DddA_1397N. The indicated peak fraction was analyzed by SDS-PAGE. b, Size-exclusion chromatography profile of the His-tagged R_TALE-DddA_1397C. The indicated peak fraction was analyzed by SDS-PAGE. c, Size-exclusion chromatography profile of the His-Sumo-tagged R_TALE-DddA_1397C-TadA8e. The indicated peak fraction was analyzed by SDS-PAGE. For a-c, the gels are representative of three independent experiments.
Extended Data Fig. 3 In vitro adenosine deamination assay for TALEDs.
a, Schematic of the constructions of R_TALE-DddA_1397C-TadA8e and the synthesized ssDNA that contains only one adenosine at position 11 (ssDNA_A11). b, In vitro adenosine deamination assay of R_TALE-DddA_1397C-TadA8e with ssDNA_A11 as the substrate. c, Schematic of the purified R_TALE-DddA_1397C-TadA8e targeting the synthesized ND1 dsDNA with the labeled bottom-strand and the top-strand that contains a tetrahydrofuran (dsDNA_BL_T#) in the absence or presence of APE1. d, In vitro adenosine deamination assay of R_TALE-DddA_1397C-TadA8e with dsDNA_BL_T# as the substrate. e, Schematic of the purified R_TALE-DddA_1397C-TadA8e targeting the synthesized ND1 dsDNA with the labeled bottom-strand that contains a single adenosine in spacer region and the top-strand that contains a fixed uridine (dsDNA_A2_BL_TU and dsDNA_A5_BL_TU). f, In vitro adenosine deamination assay of R_TALE-DddA_1397C-TadA8e with dsDNA_A2_BL_TU or dsDNA_A5_BL_TU as the substrate. The gels are representative of three independent experiments.
Extended Data Fig. 4 Roles of different DNA exonucleases in TALED-mediated A-to-G editing in mtDNA.
a, C-to-T editing frequencies induced by DdCBE at the ND1 and ND4 sites in wild-type (WT) and hUNG/hSMUG1 double-knockout (hUNG/hSMUG1_DKO) 293FT cells. b, A-to-G editing frequencies induced by sTALED at the ND1 and ND4 sites in WT and hAPE1 knockout (hAPE1_KO) 293FT cells. c, A-to-G editing frequencies induced by sTALED at the ND1 and ND4 sites in WT, hExo1 knockout (hExo1_KO), hExoG knockout (hExoG_KO) and hDNA2 knockout (hDNA2_KO) 293FT cells. d, Schematic diagram of the scenario in which hMGME1-mediated DNA strand excision destroys the binding region for TadA8e-containing half of sTALED and therefore no A-to-G editing is induced. e, Schematic diagram of the scenario in which DddA mediates C-to-U editing at both DNA strands in the spacer region and therefore DNA double strand breaks (DSBs) are generated. f, Schematic diagram of the scenario in which the previous round of A-to-G editing (red base pair) occurred in one DNA strand (dark green strand) within the spacer region and therefore a new round of A-to-G editing (orange base) can occur in the other DNA strand (light green strand). For a-c, the data are presented as the mean ± s.d. from three independent experiments.
Extended Data Fig. 5 Generation of hExoG, hMGME1 and hDNA2 knockout cells and overexpression of hUNG and hMGME1.
a, c, e, The knockouts of indicated genes were confirmed by sequencing the genomic loci. b, d, f, The effects of knocking out hExoG, hMGME1 and hDNA2 on protein expression were examined by Western blots. The blots for anti-hExoG/anti-hMGME1/anti-hDNA2 and anti-actin/anti-tubulin were performed with the same samples but run in different gels. g, The expression of myc-hUNG when editing the ND1 and ND4 sites in hUNG/hSMUG1_DKO cells was examined by Western blots. The blots for anti-myc and anti-tubulin were performed with the same samples but run in different gels. h, A-to-G editing frequencies induced by sTALED without or with myc-hUNG coexpression at the ND1 and ND4 sites in hUNG/hSMUG1_DKO cells. i, The expression of myc-hMGME1 when editing the ND1 and ND4 sites in hMGME1_KO cells was examined by Western blots. The blots for anti-myc and anti-tubulin were performed with the same samples but run in different gels. j, A-to-G editing frequencies induced by sTALED without or with myc-hMGME1 coexpression at the ND1 and ND4 sites in hMGME1_KO cells. For h and j, the data are presented as the mean ± s.d. from three independent experiments.
Extended Data Fig. 6 Comparison of the A-to-G editing and C-to-T editing induced by the TALEDs and DdCBEs with same TALE arrays.
a, Schematic of the constructions of sTALED and DdCBE targeting mitochondrial DNA. b, A-to-G and C-to-T editing frequencies induced by DdCBE and sTALED at the ND5.1, ND6.1, TRNG, ND4L and ND5.3 sites in 293FT cells. The data are presented as the mean ± s.d. from three independent experiments.
Extended Data Fig. 7 Editing efficiency induced by engineered TALEDs.
a, Schematic of the constructions of sTALED, sTALED6 and sTALED11 targeting mtDNA. b, A-to-G editing frequencies induced by sTALED, sTALED6 and sTALED11 at the ND5.1 and ND5.3 sites in 293FT cells. c, Schematic of the constructions of sTALED, esTALED_V2 and esTALED_V2_dhUNG targeting mtDNA. dhUNG, catalytically dead hUNG. d, A-to-G editing frequencies induced by sTALED, esTALED_V2 and esTALED_V2_dhUNG at the ND1 and ND5.3 sites in 293FT cells. e, Schematic of the constructions of sTALED_G1333 and esTALED_G1333 targeting mtDNA. f, A-to-G editing frequencies induced by sTALED_G1333 and esTALED_G1333 at the ND1, ND5.3 and ND5.3 sites in 293FT cells. For b, d and f, the data are presented as the mean ± s.d. from three independent experiments.
Extended Data Fig. 8 Comparison of editing efficiencies of mTALEDs and the effect of complete DddA deamination disruption on TALED efficiency.
a, Schematic of the constructions of mTALED, mTALED6_GSVG, emTALED6_GSVG_V1 and emTALED6_GSVG_V2 targeting mtDNA. b, A-to-G editing frequencies induced by mTALED, mTALED6_GSVG, emTALED6_GSVG_V1 and emTALED6_GSVG_V2 at the ND1 and ND5.1 sites in 293FT cells. c, Schematic of the constructions of mDdCBE_E1347A and mDdCBE_E1347A-G1348S targeting mtDNA. d, C-to-T editing frequencies induced by mDdCBE_E1347A and mDdCBE_E1347A-G1348S at the ND1, ND5.1 and ND5.3 sites in 293FT cells. e, Schematic of the constructions of dTALED and dTALED_E1347A-G1348S targeting mtDNA. f, A-to-G editing frequencies induced by dTALED and dTALED_E1347A-G1348S at the ND1, ND5.1 and ND5.3 sites in 293FT cells. For b, d and f, the data are presented as the mean ± s.d. from three independent experiments.
Extended Data Fig. 9 Comparison of edTALED6_GSVG with other mtDNA editors and characterization of its cell applicability and mtDNA-wide off-target editing.
a, Schematic of the construction of the dTALED, mitoABENt.BspD6I(C), monomeric mitoABENt.BspD6I(C) and edTALED6_GSVG targeting mtDNA. b, Heatmaps of A-to-G editing frequencies induced by the indicated editors. c and d, A-to-G editing frequencies induced by dTALED6_GSVG and edTALED6_GSVG in A549 (c) and U2OS (d) cells. For c and d, the data are presented as the mean ± s.d. from three independent experiments. e, The position and frequency of single base substitution in mtDNA induced by the indicated editors targeting the ND5.1 site, with the editing within the spacer region shown in red. The data are presented from three independent experiments.
Extended Data Fig. 10 Characterization of the editing efficiency, nuclear genome-wide mutation and cellular toxicity induced by engineered TALEDs.
a, Schematic of the constructions of sTALED, esTALED6 and esTALED6R targeting mtDNA. b, Heatmaps of A-to-G editing frequencies induced by the indicated editors. The sequences of the TALE-binding regions are in blue, and bystander editing can be found in the boxes with dashed black lines. The boxes with dashed blue lines represent the narrowed editing windows. c and d, Number of A/T-to-G/C (c) and C/G-to-T/A (d) nuclear genome-wide substitutions induced by the indicated editors. e and f, Cell viability was measured by MTS assay at day 2 (e) and day 4 (f) post-transfection after treated with the indicated editors targeting ND1 site. For b, e and f, the data are presented as the mean ± s.d. from three independent experiments. For c and d, the eGFP data are presented as the mean ± s.d. from two independent experiments, sTALED, esTALED6 and esTALED6R are presented as the mean ± s.d. from three independent experiments. Statistical analysis was performed via two-tailed Student’s t-test. For e and f, statistical analysis was performed via two-tailed Welch’s t-test.
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Fan, Y., Xu, W., Gao, BQ. et al. Leveraging base excision repair for efficient adenine base editing of mitochondrial DNA. Nat Biotechnol 44, 277–290 (2026). https://doi.org/10.1038/s41587-025-02608-w
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DOI: https://doi.org/10.1038/s41587-025-02608-w
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