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Mitochondrial base editor induces substantial nuclear off-target mutations

Abstract

DddA-derived cytosine base editors (DdCBEs)—which are fusions of split DddA halves and transcription activator-like effector (TALE) array proteins from bacteria—enable targeted C•G-to-T•A conversions in mitochondrial DNA1. However, their genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or TAS-independent. TAS-dependent off-target sites in the nuclear DNA are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by paired TALE proteins positioned in close proximity. TAS-independent off-target sites on nuclear DNA are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with binding sites for the transcription factor CTCF and are enriched in topologically associating domain boundaries. We engineered DdCBE to alleviate such off-target effects. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base-editing tools.

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Fig. 1: DdCBE induces abundant off-target edits in the nuclear genome.
Fig. 2: DdCBE induces one-sided, TAS-dependent nDNA off-target edits.
Fig. 3: Prevalent, non-random TAS-independent off-target sites.
Fig. 4: TAS-independent nDNA off-target sites are enriched at CTCF binding sites and TAD boundaries.
Fig. 5: DdCBE variants with improved specificity.

Data availability

All data generated for this paper have been deposited at NCBI Gene Expression Omnibus (GEO) and are available under GEO accession number GSE173859 (Detect-seq data), GSE173689 (ATAC-seq data and in situ ChIP–seq data) and GSE176089 (targeted deep sequencing data). hg38 was used as the reference genome. The Hi-C, DNase-seq, Bisulfite-seq and ChIP–seq data were downloaded from the GEO or ENCODE database; accession numbers of these public data sets are available in Supplementary Table 5.

Code availability

Detect-seq tools, including several Python scripts, were deposited on GitHub (https://github.com/menghaowei/Detect-seq). Detect-seq tools can help to perform Detect-seq analysis, including but not limited to Detect-seq signal finding, enrichment testing, off-target sites identification, TALE sequence alignment and alignment results visualization.

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Acknowledgements

We thank W. Wei for providing related plasmids; H. Cheng for sharing the antibodies for mitochondrial markers; W. Xie and X. Lu for discussion about the ChIP assay; X. Zhang and Chuyun Shao for help with ATAC-seq experiments and data processing; National Center for Protein Sciences at Peking University for assistance with FACS, imaging, sequencing, Imaris, Fragment Analyzer and Agilent 4150 TapeStation System; C. Shan, L. Fu, S. Qin and Y. Guo for assistance with immunofluorescence experiments, FACS and image processing; and G. Li and X. Zhang for assistance with NGS experiments. Bioinformatics analysis was performed on the High-Performance Computing Platform of the School of Life Sciences and High-Performance Computing Platform of the Center for Life Science. This work was supported by the National Natural Science Foundation of China (nos. 21825701, 91953201, 92153303 and 22107006), National Key R&D Program (2019YFA0110900 and 2019YFA0802200) and China Postdoctoral Science Foundation (2020M680218, 2021M700238). L. Liu was supported in part by the Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences.

Author information

Authors and Affiliations

Authors

Contributions

Z.L., H.M. and C.Y. conceived and led the project. Z.L., H.M., L.L. and C.Y. designed the experiments to investigate the DdCBE off-target effect, which were performed by Z.L. and L.L. H.M. analysed Detect-seq, in situ ChIP–seq and the downloaded public data. H.Z. analysed ATAC-seq and targeted deep sequencing data. Z.L. performed the co-immunoprecipitation and non-fixation immunofluorescence assays. L.L. conducted the cell fractionation assay and the sequential western blot and fixation-based immunofluorescence experiments. X. R. and Y. Y. assisted the experiments and data processing. Z.L. and C.Y. designed the in situ ChIP experiments with the advice of A.H. M.L. performed the in situ ChIP–seq experiments with cell samples prepared by Z.L. Z.L., H.M., L.L., H.Z. and C.Y. wrote the paper with the help of X. R. and H.W.

Corresponding author

Correspondence to Chengqi Yi.

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Peking University has filed patent applications on Detect-seq and optimized DdCBE variants described in this study, listing Z.L., H.M., Z.C.L., L.L, H.Z. and C.Y. as inventors.

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Extended data figures and tables

Extended Data Fig. 1 Workflow of Detect-seq.

Endogenous 5fdC was protected by O-ethylhydroxylamine (EtONH2). Damage repair step eliminates endogenous DNA damages including abasic sites (AP), single strand breaks (SSB), etc. Deoxyuridine (dU) generated by DdCBE in vivo was labeled by the in vitro reconstituted base excision repair (BER) reaction: UDG specifically recognizes and cleaves dU, leaving abasic sites; Endo IV removes abasic sites, leaving 3’-OH remnant; Bst DNA polymerase initiates DNA strand replacement after the 3’-OH; ligase sews the final nicks. Through the so-called “nick translation” activity of Bst polymerase during this step, biotinylated dUTPs and 5fdCTPs were incorporated 3’ to dU. Malononitrile treatment marks the incorporated 5fdCs, generating a characteristic tandem C-to-T mutation pattern to trace DdCBE edits. Biotin pulldown followed by NaOH treatment enriches DdCBE edited DNA fragments and enhances Detect-seq signals.

Extended Data Fig. 2 Comparisons of Detect-seq signals for off-target edits under two different transfection conditions.

The two conditions are: 4x105 seeded HEK293T cells on 6-well plates were transfected with 4 μg of each monomer using 12 μl Lipofectamine 2000; or, 6.4x105 cells were transfected with 3.5 μg of each monomer using 21 μl Lipofectamine LTX. The Detect-seq signals are highly consistent between the two conditions for all three DdCBEs.

Extended Data Fig. 3 Editing ratios of nuclear DNA off-target sites identified for the three L1397N-DdCBEs.

a–c, Targeted deep sequencing results for selected nuclear off-target sites of ND4-L1397N (a), ND5.1-L1397N (b) and ND6-L1397N (c). For each off-target site, the editing ratio for the highest edited cytosine is plotted (blue), and the matched ratio in untreated ctrl sample is plotted in gray.

Extended Data Fig. 4 A real-time IF staining assay using unfixed HeLa nuclei to demonstrate the nuclear localization of DdCBE.

a, Fluorescence imaging of DAPI (navy blue), HA-tagged left half (Anti-HA, orange red) and Flag-tagged right half (Anti-Flag, green) in unfixed nuclei of HeLa cells untreated or transfected with Lipofectamine LTX. Possible mitochondrial contamination was tested by MitoTracker (magenta). The images were obtained at a representative Z-axis under the same exposure condition by High Speed Spinning Disk Confocal Microscope (ANDOR). Scale bars, 3 μm. Images are representative of 3 independent biological replicates. b, c, The projected 2D fluorescence image (b) and 3D snapshot (c) of a representative nuclei from cells transfected with Lipofectamine 3000. d, e, The projected 2D fluorescence image (d) and 3D snapshot (e) of a representative nuclei from cells transfected with Lipofectamine LTX. f, Statistic diagram for 3D mean fluorescence intensity per voxel of all scanned nuclei under different treatments. The data in bf for each nucleus was obtained from z-stack images collected at 0.4 μm intervals under the same exposure condition by DeltaVision OMX SR (GE). Similar color and scale bars in a were used. HeLa cells on 6-well plates were transfected with 2 μg of each monomer using 6 μl Lipofectamine3000; or, cells were transfected with 3.5 μg of each monomer using 21 μl Lipofectamine LTX. “ND6-WT”: wild type ND6-L1397N; “ND6-(TALE-)”: ND6-L1397N architectures that deleted the TALE arrays. In f, error bars reflect the mean +/− SD; and p-values are calculated by one-side Student’s t-test.

Extended Data Fig. 5 A small portion of DdCBE is localized in the nucleus of transfected HEK293T cells.

a, Western blotting results showing the distribution of ND6-L1397N (WT) in different subcellular fractions of 2×103 HEK293T cells untreated or transfected using Lipofectamine 2000 or LTX; and the distribution of three deletion variants of ND6-L1397N in different fractions of cells transfected with LTX. “DddA-free”, “UGI-free” and “TALE-free” mean the deletion of DddA, UGI and TALE arrays from the full-length ND6-L1397N respectively. The results show that ND6-L1397N is partially localized in the chromatin fraction no matter which transfection reagent was used. The signal of the TALE-free construct in the chromatin fraction is only present when the exposure time is extended. This observation suggests that compared to DddA and UGI, the TALE arrays most strongly affect the nuclear localization. ATP5a (mitochondria), GAPDH (cytosolic) and H3 (chromatin) were chosen as compartment-specific markers, demonstrating the purity of each subcellular fraction. HA (tagged left half) and Flag (tagged right half) were used to indicate the localization of DdCBEs. Molecular weight is given in kDa; images are representative of 2 independent biological replicates; samples are derived from the same batch of experiment and gels were processed in parallel. b, Fluorescence imaging of nuclei (DAPI, blue), HA-tagged left half (Anti-HA, red), Flag-tagged right half (Anti-Flag, green) in fixed nuclei isolated from HEK293T cells untreated or transfected with ND6-L1397N (WT) using Lipofectamine 2000, or transfected with ND6-L1397N (WT), DddA-free, UGI-free and TALE-free constructs using Lipofectamine LTX. Possible mitochondrial contamination was tested by MitoTracker (magenta). The results show that a small portion of DdCBE is localized in nuclei, regardless of the transfection conditions. TALE arrays more strongly affect the nuclear localization compared with DddA and UGI. Scale bars, 5 μm for zoomed in images of TALE-free; 40 μm for all remaining images. The images were obtained under the same exposure condition and are representative of 2 independent biological replicates.

Extended Data Fig. 6 The editing spectra of DdCBE at TAS-dependent nDNA off-target sites.

a, Sequence logos for Cs with highest Detect-seq signal obtained via WebLogo using DNA sequences at TAS-dependent off-target sites of ND6-L1397N, ND5.1-L1397N and ND4-L1397N. b, Sequence logos generated from the pTBSs of ND5.1-L1397N and ND4-L1397N. Bits reflect the level of sequence conservation at a given position. c, Aggregate distribution of C·Gs with highest Detect-seq signal across the flanking region of each pTBS for TAS-dependent off-target sites of ND6-L1397N, ND5.1-L1397N and ND4-L1397N. The position of pTBS for left or right TALE proteins is shadowed. d, A schematic illustrating the editing spectra of the three L1397N DdCBEs based on the pTBS-edits distribution analysis. Counting the first base pair after the 3’ ends of pTBS as position +1. NTD, N-terminal domain; CTD, C-terminal domain.

Extended Data Fig. 7 The TALE independency of TAS-independent off-target sites validated by targeted deep sequencing.

Results of targeted deep sequencing at five representative TAS-independent off-target sites for different ND6-L1397N constructs in Fig. 2a.

Extended Data Fig. 8 Motif search result from sequences of all TAS-independent off-target sites.

The results (with a p-value < 0.05) are generated by Tomtom program with JASPAR core motif database.

Extended Data Fig. 9 Strategies to improve the specificity of DdCBE.

a, Fusing nuclear export signal (NES) sequences into the DdCBE constructs. The protein level of DdCBE in the nucleus should be decreased, and hence lower the risk of nDNA off-target editing. b, Co-expressing DddIA that fused with nuclear localization signals (NLS). DddIA is a natural immunity protein of the deaminase DddA; bpNLS-linked DddIA is supposed to antagonize the deamination activity of DdCBEs mis-localized into the nucleus. bpNLS, bipartite NLS at both the N and C termini. c, Mutating the DddAtox in the DdCBE architecture to reduce its intrinsic DNA binding affinity. Ideally, mutated deaminase would not be able to catalyze DNA substrates without the help of simultaneously stable binding of the two TALE repeats.

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Lei, Z., Meng, H., Liu, L. et al. Mitochondrial base editor induces substantial nuclear off-target mutations. Nature 606, 804–811 (2022). https://doi.org/10.1038/s41586-022-04836-5

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