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C-to-G editing generates double-strand breaks causing deletion, transversion and translocation

Nature Cell Biology volume 26, pages 294–304 (2024)Cite this article

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Abstract

Base editors (BEs) introduce base substitutions without double-strand DNA cleavage. Besides precise substitutions, BEs generate low-frequency ‘stochastic’ byproducts through unclear mechanisms. Here, we performed in-depth outcome profiling and genetic dissection, revealing that C-to-G BEs (CGBEs) generate substantial amounts of intermediate double-strand breaks (DSBs), which are at the centre of several byproducts. Imperfect DSB end-joining leads to small deletions via end-resection, templated insertions or aberrant transversions during end fill-in. Chromosomal translocations were detected between the editing target and off-targets of Cas9/deaminase origin. Genetic screenings of DNA repair factors disclosed a central role of abasic site processing in DSB formation. Shielding of abasic sites by the suicide enzyme HMCES reduced CGBE-initiated DSBs, providing an effective way to minimize DSB-triggered events without affecting substitutions. This work demonstrates that CGBEs can initiate deleterious intermediate DSBs and therefore require careful consideration for therapeutic applications, and that HMCES-aided CGBEs hold promise as safer tools.

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Fig. 1: In-depth profiles of cytidine base-editing outcomes.
Fig. 2: CGBE generates intermediate DSB at editing sites.
Fig. 3: Detection of CGBE-initiated chromosomal translocation.
Fig. 4: Genetic dissection of on-target indel formation.
Fig. 5: Molecular pathways under C-to-G transversion.
Fig. 6: Minimizing on-target indels with HMCES-aided tools.

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Data availability

BE-outcomes in 513 DNA repair gene knockouts are supplied as Supplementary Table 3. Raw high-throughput sequencing data, including BE-outcome screening, Base editing amplicon, Tn5-HTGTS, ATAC-seq and END-seq, were deposited in the SRA database (accession numbers, PRJNA839540 and PRJNA841413). Other datasets were downloaded from the SRA database (accession numbers: RNA-seq, SRR9019712, SRR9019714, SRR10251291, SRR10251292, SRR7988530, SRR7988534, SRR10590683, and SRR10590685; H3K27ac ChIP–seq, SRR14879752 and SRR14879751; H3K4me3 ChIP–seq, SRR14879757 and SRR14879751; H3K27me3 ChIP–seq, SRR11040439 and SRR11040434; H3K9me3 ChIP–seq, SRR11040453 and SRR11040444; PRO-seq, SRR7988488). Source data are provided with this paper.

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Acknowledgements

We thank L.-L. Chen, G. Xu, J. Li, L. Shen, T. Honjo, K. Yu and D. Gao for providing reagents, A. Nussenzweig for critical reading and help with END-seq, X. Xie, D. Yang, Ti. Liu, Y. Zhou, L. Shan, Z. Zhuo and S. Li for technical support. This work was supported by the National Key R&D Program of China 2023YFA0913700 (F.-L.M.), National Natural Science Foundation of China grant 32090040 (F.-L.M.), Strategic Priority Research Program of Chinese Academy of Science XDB0570000 (F.-L.M.), National Natural Science Foundation of China grant 32325019 (F.-L.M.), National Natural Science Foundation of China grant 31970880 (F.-L.M.), National Natural Science Foundation of China grant 31722020 (L.-S.Y.), National Natural Science Foundation of China grant 81861138014 (L.-S.Y.), National Natural Science Foundation of China grant 32170884 (X.L.), National Key R&D Program of China 2021YFA1301400 (L.-S.Y.). Shanghai Municipal Science and Technology Major Project HS2021SHZX001 (F.-L.M.), Natural Science Foundation of Shanghai grant 23XD1424000 (F.-L.M.), Chinese Academy of Sciences grant JCTD-2020-17 (F.-L.M. and X.Z.) and Chinese Academy of Sciences grant 318GJHZ2022010MI (F.-L.M.).

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Author notes

  1. These authors contributed equally: Min Emma Huang, Yining Qin, Yafang Shang.

Authors and Affiliations

  1. Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

    Min Emma Huang, Yafang Shang & Fei-Long Meng

  2. Key Laboratory of RNA Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of SciencesUniversity of Chinese Academy of Sciences, Shanghai, China

    Min Emma Huang, Yining Qin, Yafang Shang, Liu Daisy Liu, Shuheng Wu, Yifeng Luo, Yanni Cai, Xiaojing Liu, Ziye Xu, Pengfei Dai, Simiao Li, Wei Wu & Fei-Long Meng

  3. Department of Immunology and Microbiology, Shanghai Institute of Immunology, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Qian Hao, Chuanzong Zhan, Chaoyang Lian, Simin Luo, Tuantuan Gui, Binbin Wang & Leng-Siew Yeap

  4. Department of Endocrinology and Metabolic Diseases, Center for Immune-Related Diseases at Shanghai Institute of Immunology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Qian Hao, Chuanzong Zhan & Leng-Siew Yeap

  5. Department of Mathematics, Shanghai Normal University, Shanghai, China

    Senxin Zhang

  6. Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yu Zhang & Yingjie Xu

  7. Departments of Thoracic Surgery and State Key Laboratory of Genetic Engineering, Fudan University Shanghai Cancer Center, Institute of Thoracic Oncology, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China

    Yang Wo & Yihua Sun

  8. Department of Medical Genetics and Molecular Diagnostic Laboratory, Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Niu Li

  9. Hefei National Research Center for Cross Disciplinary Science, Ministry of Education Key Laboratory for Membraneless Organelles and Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Liang Zhang

  10. Department of Immunology and Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China

    Junchao Dong

  11. International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Jian Wang

  12. Center for Single-Cell Omics, School of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Xiaoqi Zheng

  13. Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yingjie Xu

  14. Shanghai Sci-Tech Inno Center for Infection & Immunity, Shanghai, China

    Fei-Long Meng

Authors
  1. Min Emma Huang
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  2. Yining Qin
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  3. Yafang Shang
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Contributions

F.-L.M. and L.-S.Y. developed the concept for the study. F.-L.M., L.-S.Y., W.W., Y. Sun, X.Z. and Y.X. developed the methodology. M.E.H., Y.Q., Y. Shang, Q.H., C.Z., C.L., S.L., L.D.L., S.Z., Y.Z., Y.W., N.L., S.W., T.G., B.W., Y.L., Y.C., X.L., Z.X., S.L. and P.D. performed the investigations. N.L., J.W., Y.W., Y. Sun, L.Z. and J.D. provided resources. M.E.H., Y.Q., Y.Shang, F.L.M. and L.S.Y. worked on visualization. F.L.M., L.S.Y., X.L. and X.Z. sourced funding. F.-L.M., L.S.Y., W.W., Y. Sun, Y.X., X.Z. and J.W. F.-L.M. and L.-S.Y. wrote the draft paper. F.-L.M., L.S.Y., W.W., Y. Sun, Y.X., X.Z., J.W., M.E.H., Y.Q. and Y. Shang reviewed and edited the final paper.

Corresponding authors

Correspondence to Leng-Siew Yeap or Fei-Long Meng.

Ethics declarations

Competing interests

Shanghai Institute of Biochemistry and Cell Biology has filed a patent application based on the findings in this article. Application number, 2022109688505; Inventor initials, F.-L.M., M.H., Y.C., Y.Q., Y. Shang, L.-D.L., X.L.

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Extended data

Extended Data Fig. 1 Cytidine editing outcome in-depth analyses.

a, Schematic illustration of the cytidine editing tools tested in the in-depth profiling assay. b, Substitution frequency (top) and C > G purity (bottom) of the indicated tools at 4 sgRNA targeting loci. Mean ± SEM of three biological replicates is shown. c, On-target deletion frequency at the 4 loci. Mean ± SEM of three biological replicates is shown. d, Representative western blot of BE expression from three replicates. e, Size distribution of on-target deletions. Mean ± SEM of three biological independent replicates are shown. f, Substitution and deletion profiles at the indicated loci after editing. Panel is labeled as Fig. 1f. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 2 Cytidine editing initiates on-target deletion in different cell lines and sgRNA targets.

a, Substitution frequency and deletion profile are shown at the indicated loci upon cytidine editing in human dermal fibroblast and Hela cells. b, Deletion frequency is plotted against substitution frequency at the indicated locus across different cell types. Linear regression analysis was performed and Pearson’s r is shown. The significance of regression model was calculated by the F-test. Two-tailed p-value is shown. c, On-target deletion frequencies at 18 tested sgRNA target loci. Data from one replicate are shown. For boxplot, centre line indicates the median; box limits represent the upper and lower quartiles, and the whiskers represent minimum to maximum values. Source numerical data are available in source data.

Source data

Extended Data Fig. 3 Insertion profiles generated by BEs.

a, Insertion profiles are shown at the indicated loci after cytidine editing. 1bp-insertion are shown at the top and >1bp-insertion are shown at the bottom for each locus. In the profile, the inserted position was counted and the frequency is plotted along the nucleotides. b, Insertion subgroups. 1-bp insertions at a mononucleotide track (“Mono”), editing sites (“Edit”), or the Cas9-nicking site (“Nick”), and >1 bp insertions with duplication sequences are shown at the top. Example insertions are labelled along the DNA sequences. The count number of a particular insertion is shown in different colors from the total 1.9 million sequencing reads. c, Substitution, deletion and >1 bp insertion frequencies are summarized for the indicated tools at 4 tested loci from three biological replicates. For the Cas9 module, dCas9 (d) or nCas9 (n) was tested; for the AID/APOBEC module, catalytically active (+) or dead (-) module was tested. Two-tailed Student’s paired t-test was applied. For boxplot, centre line indicates the median; box limits represent the upper and lower quartiles, and the whiskers represent minimum to maximum values. Source numerical data are available in source data.

Source data

Extended Data Fig. 4 Intermediate DSBs revealed by different approaches.

a, Deletions and insertions at 11 loci after CGBE editing. Mean ± SD of three replicates is shown. Two-tailed Student’s paired t-test was applied. b, Knockout of XRCC4 was validated by Western blot (left). Expression of CGBE1 was examined in the indicated cell lines (right). Representative blots from two replicates are shown. c, Illustration of the END-seq experiment procedure. d, Expression levels of CGBE1, nCas9, and Cas9 are shown by a representative Western blot from two replicates. e, Two additional replicates of END-seq are shown. Panel is labeled as in Fig. 2d. f, Immunofluorescence for γH2AX in WT and XRCC4-deficient HEK293T cells with or without AID-CGBE editing. The sgRNA targeting a sgEMX1 site was used. One representative replicate is shown. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 5 Chromosomal translocation profiles.

a, Circos plots of genome-wide translocation junctions in cells treated with nCas9 or the indicated BEs. Translocation junctions were binned into 5-Mb regions (black bars) and plotted on a log scale; the orange line connects the on-target bait site with a Cas-OT prey hotspot, the green line connects the on-target bait site with a deaminase-OT prey hotspot. The middle number indicates unique translocation junctions from 3.6 million edited cells. b, Distribution of translocation junctions at Cas-OT hotspots of an sgEMX1 on-target bait in cells edited with the indicated BE. c, Translocation, H3K27Ac and transcription profiles at an AID deaminase-OT hotspot. Top: Translocation junctions are indicated by black bars from cells transfected with AID-CGBE or CGBE1. Middle (H3K27Ac and SE): The H3K27Ac ChIP-seq profile is shown in orange, and identified SEs are depicted below with orange bars. Bottom (PRO-seq): PRO-seq detected sense and antisense transcription are shown in blue and red, respectively. d, Enrichment of scattered translocation junctions at genomic regions associated with the indicated histone markers, open chromatin and transcription in cells transfected with AID-CGBE from the 4 baits. Each dot indicates average number of two biological replicates at the indicated locus. Mean ± SD of 4 sgRNA loci is shown. e, Relative motif enrichment in AID-CGBE-edited samples compared to that of random sampling of the human genome. Each dot indicates average number of two biological replicates at the indicated locus. Mean ± SD of 4 sgRNA loci is shown. f, Substitution frequency (left) and C > G purity (right) of indicated pairs of synthetic sequences with and without palindromic deamination motif. The editing assay was performed in a 48-well plate format. Four independent replicates are shown. For boxplot, centre line indicates the median; box limits represent the upper and lower quartiles, and the whiskers represent minimum to maximum values. g, Substitution (upper) and deletion (lower) profile of CGBE1 on 5 pairs of synthetic sequences as shown in Fig. 1f. “TCAA” was applied as a control for “TCGA”. h, Percentage of sgRNAs containing palindromic motifs in the editing window. A total of 3040 potential CGBE-correcting loci were analysed. For Panels d and e, two-tailed one-sample t-test was performed. For Panel f, two-tailed Student’s paired t-test was applied. Source numerical data are available in source data.

Source data

Extended Data Fig. 6 BE outcome-screening.

a, Genes in the DDR-focused library, the Repair-seq library of Koblan et al., 2021, and the core DNA repair gene list defined by Olivieri et al, 2020, are shown in a Venn diagram with the numbers of genes labeled. b, Schematic illustration of the SaBE tools used in the CRISPR screening assay, including SaCGBE1, AID-SaBE3, AID-SaCGBE, and AID-SaBE1. c, Substitution profiles of the SaBE-Test locus for SaCGBE1 from 6 million reads. Deletion boundary profiles are shown at right. d, Substitution profiles of the SaBE-Test for AID-SaBE3 or AID-SaBE1 from 6 million reads. Deletion boundary profiles are shown at right. e, Schematic illustration of the BE outcome groups, with the example sequences of each group shown on the right. f, Radar plots showing base editing outcomes of gene knockouts. The wild-type result was used as a reference (blue line) in each panel. Source numerical data are available in source data.

Source data

Extended Data Fig. 7 CGBE editing outcomes in DNA repair deficient cell lines.

a, Deletion, substitution frequencies, and C > G purity are plotted for the indicated loci. Mean ± SD of 4 replicates is shown in the bar graph. The expression levels of indicated tools are shown by a representative Western blot on the right. b, Deletion (left) and substitution (right) frequencies were plotted for the 3 indicated loci in the indicated cell lines. Mean ± SD of three replicates is shown in the bar graph. Expression levels of CGBE1 in the indicated cells are shown by a representative Western blot from two biological replicates. A 3-fold serial dilution of protein samples was applied in Western blot. c, MH usage in the deletion junctions at 2 loci in the indicated mouse B cell lines. Mean ± SD of 3 replicates is shown. Two-tailed Student’s paired t-test was applied. d, Fractions of insertions are plotted for the indicated tools. e, Proportions of 1-bp and >1 bp insertions, and proportions of duplication and other insertions of >1 bp insertions are shown. f, Insertions at the SaBE test locus. Four insertion subtypes are shown at the top and example duplications are illustrates at the bottom. g, Negatively-enriched genes in >1 bp insertions. The heatmap is shown as in Fig. 4c. The p-value was also calculated by permutation test and FDR was calculated by the Benjamini-Hochberg procedure from MAGeCK. h, 1 bp and >1 bp insertion profiles of HEK293T XRCC4-/- and its parental cell lines at the sgFANCF locus. i, Expression levels of CGBE1 in the indicated cells are shown by a representative Western blot from two biological replicates. A 3-fold serial dilution of protein samples was applied in Western blot. j, Substitution, C > G purity, >1 bp insertion, and deletion are plotted. Mean ± SD of three replicates. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 8 DSB end-joining induces aberrant C > G transversion.

a, C > G purity at three loci in CasRx-knockdown HEK293T cells. Each dot indicates average C > G purity of each positions from three biological replicates. The RFWD3 mRNA levels are shown on the left (mean ± SD of 3 biological replicates). b, Genotoxicity of REV1-deficient HEK293T cells after UV treatment. Mean ± SD of 3 replicates is shown. Knockout of REV1 was validated by Western blot. Representative blot is shown from 2 replicates. c, C > G purity after CGBE editing is plotted for each locus. Mean ± SD of 3 replicates is shown. Expression levels of CGBE1 in the indicated cells are shown by a representative Western blot from 2 replicates. A 3-fold serial dilution of protein samples was applied in Western blot. d, C > G purity is plotted for 3 loci in the Rev1-/-, Rev7-/-, Rev3l-/- and parental CH12F3 cell line. Mean ± SD of 3 independent replicates is shown. e, Expression levels of CGBE1 in the indicated cells are shown by a representative Western blot. A 3-fold serial dilution of protein samples was applied in Western blot. f, C > G purity after CGBE editing is plotted for each locus. Mean ± SD of 3 replicates is shown. g, Fold change of C > G purity in XRCC4 deficiency vs. wild-type is plotted against that of deletion frequency at 11 sgRNA loci. Linear regression analysis was performed and Pearson’s r is shown. The significance of regression model was calculated by the F-test. h, A working model of C > G transversion. For Panels a, c and f, two-tailed Student’s paired t-test was applied. For Panel b, two-tailed Student’s unpaired t-test was applied. For boxplots in a and c, centre line indicates the median; box limits represent the upper and lower quartiles, and the whiskers represent minimum to maximum values. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 9 Optimization and characterization of CGBEfH/pH tools.

a, A schematic of the AID-CGBEfH1 construct is shown on top. Deletion frequencies at three sgRNA loci from three biological replicates are plotted at bottom. The same sgRNA loci are connected by a line. Mean ± SD is shown. b, Relative deletion frequency is shown as a heatmap for the AID-CGBEfH2 at sgFANCF locus. Deletion frequency was compared to that generated by AID-CGBE. c, Schematic illustration of C > G screening and protein evolution strategy. Validation of the mutations is shown on the right. Deletion and substitution frequencies are shown for each tool. Two biological replicates are shown for K83R/E165A/P185E/Q235K/Q235R. For other mutants, mean ± SD of 3 biological replicates is shown. For boxplots, centre line indicates the median; box limits represent the upper and lower quartiles, and the whiskers represent minimum to maximum values. d, Deletion and >1 bp insertion frequencies are plotted at the 7 sgRNA loci tested for the indicated tools. Mean ± SD of 3 biological replicates. Expression of CGBE tools is shown by Western blot. Representative blot is shown from 2 replicates. e, >1 bp insertion frequency is plotted at the 7 sgRNA loci tested for the indicated tools. Mean ± SD of 3 replicates. Expression of CGBE and HMCES is shown by Western blot. Representative blot is shown from 2 replicates. f, Deletion and C > G frequency are shown as mean ± SD of 3 biological replicates. g, C > G frequency are shown for synthetic ‘TCGA’ sgRNA sequences. Mean ± SD of 4 biological replicates. h, Deletion frequency is shown as mean ± SD of 3 replicates for the indicated tools. i, Numbers of normalized END-seq DSB ends are plotted along a 50 bp region of the sgEMX1 or sgFANCF locus. The orientation of DSB ends is indicated in blue (+) or red (-). j, Translocation junctions upon CGBE1 or CGBE1pH editing from the sgHBB bait. For Panels a, c, d, e, f, g and h, two-tailed Student’s paired t-test was applied. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 10 High-fidelity Cas or optimized deaminase module cannot reduce CGBE-initiated deletion and GFP gating strategy.

a, CGBE1-derived tools with modified Cas/deaminase modules are shown on the top. Substitution frequency, C > G purity, deletion frequency, and insertion frequency were shown side-by-side between CGBE1 and optimized tool at 4 tested loci. Data from two independent replicates are shown. Source numerical data are available in source data. b, GFP gating strategy. In the transfection efficiency control assay, cells co-transfected with a GFP-expressing plasmid were gated on FSC and SSC for live cells. Live cells were further gated on FITC intensity. Non-transfected cells were assayed as a negative control.

Source data

Supplementary information

Reporting Summary

Peer Review File

Supplementary Table 1

Detail data of Tn5-HTGTS and END-seq.

Supplementary Table 2

BE outcome-screening results.

Supplementary Table 3

Editing efficiency, substitution spectrum and indel frequency in 513 DNA repair deficiencies.

Supplementary Table 4

Primers, plasmids and antibodies used in this study.

Source data

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Huang, M.E., Qin, Y., Shang, Y. et al. C-to-G editing generates double-strand breaks causing deletion, transversion and translocation. Nat Cell Biol 26, 294–304 (2024). https://doi.org/10.1038/s41556-023-01342-2

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