Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Strand-preferred base editing of organellar and nuclear genomes using CyDENT

Nature Biotechnology (2023)Cite this article

Subjects

Abstract

Transcription-activator-like effector (TALE)-based tools for base editing of nuclear and organellar DNA rely on double-stranded DNA deaminases, which edit substrate bases on both strands of DNA, reducing editing precision. Here, we present CyDENT base editing, a CRISPR-free, strand-selective, modular base editor. CyDENT comprises a pair of TALEs fused with a FokI nickase, a single-strand-specific cytidine deaminase and an exonuclease to generate a single-stranded DNA substrate for deamination. We demonstrate effective base editing in nuclear, mitochondrial and chloroplast genomes. At certain mitochondrial sites, we show editing efficiencies of 14% and strand specificity of 95%. Furthermore, by exchanging the CyDENT deaminase with one that prefers editing GC motifs, we demonstrate up to 20% mitochondrial base editing at sites that are otherwise inaccessible to editing by other methods. The modular nature of CyDENT enables a suite of bespoke base editors for various applications.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Strand-selective base editing using CyDENT in plant cells.
Fig. 2: Strand-selective mtDNA base editing using CyDENT in HEK293T cells.
Fig. 3: Optimization of CyDENT for mtDNA base editing.
Fig. 4: mtCyDENT enables efficient strand-selective base editing under GC contexts.
Fig. 5: Whole nuclear genome and mitochondrial genome-wide off-target analysis of mtCyDENT.

Similar content being viewed by others

Data availability

The deep amplicon sequencing data are deposited at the NCBI as Bioproject PRJNA957099 and PRJNA957096 ref. 54. All other data are available in the paper or Supplementary Information.

References

  1. Boore, J. L. Animal mitochondrial genomes. Nucleic Acids Res. 27, 1767–1780 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Tuppen, H. A., Blakely, E. L., Turnbull, D. M. & Taylor, R. W. Mitochondrial DNA mutations and human disease. Biochim. Biophys. Acta 1797, 113–128 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Greaves, L. C., Reeve, A. K., Taylor, R. W. & Turnbull, D. M. Mitochondrial DNA and disease. J. Pathol. 226, 274–286 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR–Cas system. Nat. Biotechnol. 31, 686–688 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438–440 (2017).

    Article  CAS  PubMed  Google Scholar 

  10. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).

    Article  CAS  PubMed  Google Scholar 

  12. Yarnall, M. T. N. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat. Biotechnol. 41, 500–512 (2023).

    Article  CAS  PubMed  Google Scholar 

  13. Sun, C. et al. Precise integration of large DNA sequences in plant genomes using PrimeRoot editors. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01769-w (2023).

    Article  PubMed  Google Scholar 

  14. Mok, B. Y. et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583, 631–637 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lee, S., Lee, H., Baek, G. & Kim, J. S. Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors. Nat. Biotechnol. 41, 378–386 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Kang, B. C. et al. Chloroplast and mitochondrial DNA editing in plants. Nat. Plants 7, 899–905 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lei, Z. et al. Mitochondrial base editor induces substantial nuclear off-target mutations. Nature 606, 804–811 (2022).

    Article  CAS  PubMed  Google Scholar 

  18. Cho, S. I. et al. Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases. Cell 185, 1764–1776 (2022).

    Article  CAS  PubMed  Google Scholar 

  19. Yin, L., Shi, K. & Aihara, H. Structural basis of sequence-specific deamination by double-stranded DNA deaminase toxin DddA. Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-023-01034-3 (2023).

    Article  PubMed  Google Scholar 

  20. Wah, D. A., Hirsch, J. A., Dorner, L. F., Schildkraut, I. & Aggarwal, A. K. Structure of the multimodular endonuclease FokI bound to DNA. Nature 388, 97–100 (1997).

    Article  CAS  PubMed  Google Scholar 

  21. Bitinaite, J., Wah, D. A., Aggarwal, A. K. & Schildkraut, I. FokI dimerization is required for DNA cleavage. Proc. Natl Acad. Sci. USA 95, 10570–10575 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Miller, J. C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Szczepek, M. et al. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat. Biotechnol. 25, 786–793 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Waugh, D. S. & Sauer, R. T. Single amino acid substitutions uncouple the DNA binding and strand scission activities of Fok I endonuclease. Proc. Natl Acad. Sci. USA 90, 9596–9600 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lee, B. I., Shannon, M., Stubbs, L. & Wilson, D. M. 3rd Expression specificity of the mouse exonuclease 1 (mExo1) gene. Nucleic Acids Res. 27, 4114–4120 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Certo, M. T. et al. Coupling endonucleases with DNA end-processing enzymes to drive gene disruption. Nat. Methods 9, 973–975 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Chen, M. J., Ma, S. M., Dumitrache, L. C. & Hasty, P. Biochemical and cellular characteristics of the 3′ → 5′ exonuclease TREX2. Nucleic Acids Res. 35, 2682–2694 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).

    Article  CAS  Google Scholar 

  29. Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sanders, K. L., Catto, L. E., Bellamy, S. R. & Halford, S. E. Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands. Nucleic Acids Res. 37, 2105–2115 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Li, R. et al. High-efficiency plastome base-editing in rice with TAL cytosine deaminase. Mol. Plant 14, 1412–1414 (2021).

    Article  CAS  PubMed  Google Scholar 

  32. Nakazato, I. et al. Targeted base editing in the plastid genome of Arabidopsis thaliana. Nat. Plants 7, 906–913 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jiang, Z., Yang, M., Zhang, Y., Jackson, A. O. & Li, D. In Encyclopedia of Virology 4th edn, Vol. 3 (eds Bamford, D. H. & Zickerman, M.) 420–429 (Academic Press, 2021).

  34. Bonillo, M., Pfromm, J. & Fischer, M. D. Challenges to gene editing approaches in the retina. Klin. Monbl. Augenheilkd. 239, 275–283 (2022).

    Article  PubMed  Google Scholar 

  35. Huang, J. et al. Discovery of deaminase functions by structure-based protein clustering. Cell 186, 3182–3195 (2023).

    Article  CAS  PubMed  Google Scholar 

  36. Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kim, Y. et al. A library of TAL effector nucleases spanning the human genome. Nat. Biotechnol. 31, 251–258 (2013).

    Article  CAS  PubMed  Google Scholar 

  38. Nakazato, I. et al. Targeted base editing in the mitochondrial genome of Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 119, e2121177119 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Mok, B. Y. et al. CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA. Nat. Biotechnol. 40, 1378–1387 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Mi, L. et al. DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing. Nat. Commun. 14, 874 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Shimura, M. et al. Development of Leigh syndrome with a high probability of cardiac manifestations in infantile-onset patients with m.14453G > A. Mitochondrion 63, 1–8 (2022).

    Article  CAS  PubMed  Google Scholar 

  42. Wei, Y. et al. Mitochondrial base editor DdCBE causes substantial DNA off-target editing in nuclear genome of embryos. Cell Discov. 8, 27 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Yi, Z. et al. Strand-selective base editing of human mitochondrial DNA using mitoBEs. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01791-y (2023).

  46. Shan, Q. et al. Rapid and efficient gene modification in rice and Brachypodium using TALENs. Mol. Plant 6, 1365–1368 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Jin, S., Lin, Q., Gao, Q. & Gao, C. Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs). Nat. Protoc. 18, 831–853 (2023).

    Article  CAS  PubMed  Google Scholar 

  48. Chen, S. Ultrafast one‐pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta 2, e107 (2023).

    Article  Google Scholar 

  49. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Tarasov, A., Vilella, A. J., Cuppen, E., Nijman, I. J. & Prins, P. Sambamba: fast processing of NGS alignment formats. Bioinformatics 31, 2032–2034 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Van der Auwera, G. A. et al. From FastQ data to high confidence variant calls: the genome analysis toolkit best practices pipeline. Curr. Protoc. Bioinformatics 43, 11.10.1–11.10.33 (2013).

    PubMed  Google Scholar 

  52. Danecek, P. et al. Twelve years of SAMtools and BCFtools. Gigascience 10, 1–4 (2021).

  53. Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

  54. Hu, J. et al. Strand-selective base editing of organellar and nuclear genomes with TALE fusions. National Center for Biotechnology Information (NCBI) https://www.ncbi.nlm.nih.gov/bioproject/PRJNA957096 (2023).

Download references

Acknowledgements

We thank Y. Ai for kindly providing the HEK293T cell line. We also thank C. Yi for providing DdCBE vectors targeting ND1, ND4, ND5.1 and ND6. We are grateful to Y. Wang for the helpful discussions. We acknowledge Y. Li for assistance with figure drawing. We thank J. Huang at BGI Genomics for the whole-genome sequencing service. This work was supported by the Ministry of Agriculture and Rural Affairs of China, the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020102), the National Key Research and Development Program (2022YFF1002802) and the National Natural Science Foundation of China (32388201).

Author information

Author notes

  1. These authors contributed equally: Jiacheng Hu, Yu Sun, Boshu Li.

Authors and Affiliations

  1. State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Jiacheng Hu, Yu Sun, Boshu Li, Guanwen Liu & Caixia Gao

  2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

    Yu Sun, Boshu Li & Caixia Gao

  3. Qi Biodesign, Beijing, China

    Zhen Liu, Zhiwei Wang, Qiang Gao, Mengyue Guo & Kevin Tianmeng Zhao

Authors
  1. Jiacheng Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yu Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Boshu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhen Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhiwei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qiang Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mengyue Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Guanwen Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Kevin Tianmeng Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Caixia Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.H., K.T.Z. and C.G. conceived the project and designed the experiments; J.H. designed vectors and tested mtCyDENT in HEK293T cells; Y.S. and B.L. designed vectors and tested nuCyDENT and cpCyDENT in plants; J.H. and Y.S. collected and analyzed MiSeq data; J.H., Y.S. and G.L. prepared MiSeq samples; Z.L. and M.G. prepared plasmids and HEK293T cells; Z.W. and Q.G. wrote scripts and processed the MiSeq RAW data; Q.G. processed the whole-genome sequencing RAW data; J.H., Y.S. and B.L. prepared the figures; J.H., Y.S., B.L., K.T.Z. and C.G. wrote the manuscript with input from all authors; and C.G. and K.T.Z. supervised the study.

Corresponding authors

Correspondence to Kevin Tianmeng Zhao or Caixia Gao.

Ethics declarations

Competing interests

The authors have submitted a patent application based on the results reported in this paper. K.T.Z. is a founder and employee at Qi Biodesign. Z.L., Z.W., Q.G. and M.G. are employees of Qi Biodesign.

Peer review

Peer review information

Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, Supplementary Tables 1–5, Supplementary Code, Supplementary Sequences and Supplementary Refs.

Reporting Summary

Rights and permissions

Springer 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 Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hu, J., Sun, Y., Li, B. et al. Strand-preferred base editing of organellar and nuclear genomes using CyDENT. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01910-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41587-023-01910-9

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research