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.

  • Article
  • Published:

Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos

Nature Biotechnology volume 42, pages 638–650 (2024)Cite this article

Subjects

An Author Correction to this article was published on 24 April 2024

This article has been updated

Abstract

Base editors have substantial promise in basic research and as therapeutic agents for the correction of pathogenic mutations. The development of adenine transversion editors has posed a particular challenge. Here we report a class of base editors that enable efficient adenine transversion, including precise A•T-to-C•G editing. We found that a fusion of mouse alkyladenine DNA glycosylase (mAAG) with nickase Cas9 and deaminase TadA-8e catalyzed adenosine transversion in specific sequence contexts. Laboratory evolution of mAAG significantly increased A-to-C/T conversion efficiency up to 73% and expanded the targeting scope. Further engineering yielded adenine-to-cytosine base editors (ACBEs), including a high-accuracy ACBE-Q variant, that precisely install A-to-C transversions with minimal Cas9-independent off-targeting effects. ACBEs mediated high-efficiency installation or correction of five pathogenic mutations in mouse embryos and human cell lines. Founder mice showed 44–56% average A-to-C edits and allelic frequencies of up to 100%. Adenosine transversion editors substantially expand the capabilities and possible applications of base editing technology.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: 3-methyladenine DNA glycosylase-derived base editors mediate programmable adenine transversions.
Fig. 2: Characterization of AXBE in vivo and in vitro.
Fig. 3: Off-target assessment of AXBE.
Fig. 4: Evaluation of evolved mAAG in vivo and vitro.
Fig. 5: Enhanced editing properties of ACBEs via Cas embedding or TadA-8e engineering.
Fig. 6: Applications of ACBEs in mouse embryos and human cells.

Similar content being viewed by others

Data availability

HTS data have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive under accession codes PRJNA954164, PRJNA954271 and PRJNA954456 (refs. 53,54,55). RNA sequencing data have been deposited in the NCBI Sequence Read Archive under accession code PRJNA954055 (ref. 56). Source data for Figs. 1–6 and Supplementary Figs. 1–15 are presented with the paper. There are no restrictions on data availability. Source data are provided with this paper.

Change history

References

  1. Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 44, D862–D868 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. Liu, G., Lin, Q., Jin, S. & Gao, C. The CRISPR–Cas toolbox and gene editing technologies. Mol. Cell 82, 333–347 (2022).

    Article  CAS  PubMed  Google Scholar 

  3. Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).

    Article  CAS  PubMed  Google Scholar 

  4. Petri, K. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat. Biotechnol. 40, 189–193 (2022).

    Article  CAS  PubMed  Google Scholar 

  5. Liu, Y. et al. Efficient generation of mouse models with the prime editing system. Cell Discov. 6, 27 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bock, D. et al. In vivo prime editing of a metabolic liver disease in mice. Sci. Transl. Med. 14, eabl9238 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2022).

    Article  CAS  PubMed  Google Scholar 

  8. Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 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 

  10. Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci. Adv. 3, eaao4774 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. 39, 41–46 (2021).

    Article  CAS  PubMed  Google Scholar 

  12. Zhao, D. et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat. Biotechnol. 39, 35–40 (2021).

    Article  CAS  PubMed  Google Scholar 

  13. Koblan, L. W. et al. Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning. Nat. Biotechnol. 39, 1414–1425 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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 

  15. Arbab, M. et al. Determinants of base editing outcomes from target library analysis and machine learning. Cell 182, 463–480 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Caldecott, K. W. Single-strand break repair and genetic disease. Nat. Rev. Genet. 9, 619–631 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Thompson, P. S. & Cortez, D. New insights into abasic site repair and tolerance. DNA Repair (Amst). 90, 102866 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Alseth, I., Dalhus, B. & Bjoras, M. Inosine in DNA and RNA. Curr. Opin. Genet. Dev. 26, 116–123 (2014).

    Article  CAS  PubMed  Google Scholar 

  19. Schouten, K. A. & Weiss, B. Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid. Mutat. Res. 435, 245–254 (1999).

    Article  CAS  PubMed  Google Scholar 

  20. Saparbaev, M. & Laval, J. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases. Proc. Natl Acad. Sci. USA. 91, 5873–5877 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Saparbaev, M., Mani, J. C. & Laval, J. Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues. Nucleic Acids Res. 28, 1332–1339 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wyatt, M. D. & Samson, L. D. Influence of DNA structure on hypoxanthine and 1,N6-ethenoadenine removal by murine 3-methyladenine DNA glycosylase. Carcinogenesis 21, 901–908 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Lee, H. W., Dominy, B. N. & Cao, W. New family of deamination repair enzymes in uracil-DNA glycosylase superfamily. J. Biol. Chem. 286, 31282–31287 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ayala-Garcia, V. M., Valenzuela-Garcia, L. I., Setlow, P. & Pedraza-Reyes, M. Aag hypoxanthine-DNA glycosylase is synthesized in the forespore compartment and involved in counteracting the genotoxic and mutagenic effects of hypoxanthine and alkylated bases in DNA during Bacillus subtilis sporulation. J. Bacteriol. 198, 3345–3354 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sidorkina, O., Saparbaev, M. & Laval, J. Effects of nitrous acid treatment on the survival and mutagenesis of Escherichia coli cells lacking base excision repair (hypoxanthine–DNA glycosylase–ALK A protein) and/or nucleotide excision repair. Mutagenesis 12, 23–28 (1997).

    Article  CAS  PubMed  Google Scholar 

  26. Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Doman, J. L., Raguram, A., Newby, G. A. & Liu, D. R. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat. Biotechnol. 38, 620–628 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Lau, A. Y., Wyatt, M. D., Glassner, B. J., Samson, L. D. & Ellenberger, T. Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG. Proc. Natl Acad. Sci. USA. 97, 13573–13578 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lau, A. Y., Scharer, O. D., Samson, L., Verdine, G. L. & Ellenberger, T. Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision. Cell 95, 249–258 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Nishimasu, H. et al. Engineered CRISPR–Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR–Cas9 variants. Science 368, 290–296 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Li, S. et al. Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination. Nat. Commun. 11, 5827 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Chen, L. et al. Engineering a precise adenine base editor with minimal bystander editing. Nat. Chem. Biol. 19, 101–110 (2023).

    Article  CAS  PubMed  Google Scholar 

  35. 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 

  36. Tong, H. et al. Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase. Nat. Biotechnol. 1–5 (2023).

  37. Kabahuma, R. I. et al. Spectrum of MYO7A mutations in an indigenous South African population further elucidates the nonsyndromic autosomal recessive phenotype of DFNB2 to include both homozygous and compound heterozygous mutations. Genes 12, 274 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Holland, S. M. et al. STAT3 mutations in the hyper-IgE syndrome. N. Engl. J. Med. 357, 1608–1619 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Park, J. C. et al. High expression of uracil DNA glycosylase determines C to T substitution in human pluripotent stem cells. Mol. Ther. Nucleic Acids 27, 175–183 (2022).

    Article  CAS  PubMed  Google Scholar 

  40. Zeng, D. et al. Exploring C-to-G and A-to-Y base editing in rice by using new vector tools. Int. J. Mol. Sci. 23, 7990 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Zhang, X. et al. Dual base editor catalyzes both cytosine and adenine base conversions in human cells. Nat. Biotechnol. 38, 856–860 (2020).

    Article  CAS  PubMed  Google Scholar 

  42. Hamman, K. et al. Low therapeutic threshold for hepatocyte replacement in murine phenylketonuria. Mol. Ther. 12, 337–344 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Yin, S. et al. Enhanced genome editing to ameliorate a genetic metabolic liver disease through co-delivery of adeno-associated virus receptor. Sci. China Life Sci. 65, 718–730 (2022).

    Article  CAS  PubMed  Google Scholar 

  44. Yang, Y. et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat. Biotechnol. 34, 334–338 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Guan, Y. et al. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol. Med. 8, 477–488 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Chen, L. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. Nat. Biotechnol. 41, 1–10 (2022).

    Google Scholar 

  48. Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275–278 (2019).

    Article  CAS  PubMed  Google Scholar 

  49. Li, D. et al. Heritable gene targeting in the mouse and rat using a CRISPR–Cas system. Nat. Biotechnol. 31, 681–683 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Zhang, X. et al. Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain. Nat. Cell Biol. 22, 740–750 (2020).

    Article  CAS  PubMed  Google Scholar 

  51. Hwang, G. H. et al. Web-based design and analysis tools for CRISPR base editing. BMC Bioinformatics 19, 542 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Chen, L. et al. Adenine transversion editors enable precise and efficient A•T-to-C•G base editing in mammalian cells and embryos. National Center for Biotechnology Information Sequence Read Archive, BioProject PRJNA954164 (2023).

  54. Chen, L. et al. Adenine transversion editors enable precise and efficient A•T-to-C•G base editing in mammalian cells and embryos. National Center for Biotechnology Information Sequence Read Archive, BioProject PRJNA954271 (2023).

  55. Chen, L. et al. Adenine transversion editors enable precise and efficient A•T-to-C•G base editing in mammalian cells and embryos. National Center for Biotechnology Information Sequence Read Archive, BioProject PRJNA954456 (2023).

  56. Chen, L. et al. Adenine transversion editors enable precise and efficient A•T-to-C•G base editing in mammalian cells and embryos. National Center for Biotechnology Information Sequence Read Archive, BioProject PRJNA954055 (2023).

Download references

Acknowledgements

We are grateful to S. Siwko (Texas A&M University Health Science Center) for proofreading the manuscript and to support from the East China Normal University Public Platform for Innovation (011). We thank Y. Zhang from the Flow Cytometry Core Facility of the School of Life Sciences at East China Normal University and H. Jiang from the Core Facility and Technical Service Center of the School of Life Sciences and Biotechnology at Shanghai Jiao Tong University. We thank L. Ji (MedSci) for designing schematic diagrams. This work was partially supported by grants from the National Key R&D Program of China (2019YFA0110802 to D.L. and 2022YFC3400203 to Y.G.); the National Natural Science Foundation of China (32025023 and 32230064 to D.L. and 82100773 to Y.G.); the Shanghai Municipal Commission for Science and Technology (21JC1402200, 20140900200 and 20MC1920400 to D.L.); the Innovation Program of the Shanghai Municipal Education Commission (2019-01-07-00-05-E00054 to D.L. and NK2022010207 to D.L.); the Innovative Research Team of High-Level Local Universities in Shanghai (SHSMU-ZDCX20212200 to D.L.); Fundamental Research Funds for the Central Universities; and the East China Normal University Outstanding Doctoral Students Academic Innovation Ability Improvement Project (YBNLTS2021-026 to L.C.). P.B.R., A.A.S. and D.R.L acknowledge support from US National Institutes of Health grants (U01AI142756, R35GM118062 and RM1HG009490 to D.R.L) and the Howard Hughes Medical Institute.

Author information

Author notes

  1. These authors contributed equally: Liang Chen, Mengjia Hong, Changming Luan.

Authors and Affiliations

  1. Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China

    Liang Chen, Mengjia Hong, Changming Luan, Hongyi Gao, Gaomeng Ru, Xinyuan Guo, Dujuan Zhang, Shun Zhang, Jun Wu, Chao Qu, Yifan Zhu, Yuting Guan, Liren Wang, Mingyao Liu, Gaojie Song & Dali Li

  2. Department of Orthopedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases with Integrated Chinese-Western Medicine, Shanghai Institute of Traumatology and Orthopedics, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

    Changwei Li

  3. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Peyton B. Randolph, Alexander A. Sousa & David R. Liu

  4. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Peyton B. Randolph, Alexander A. Sousa & David R. Liu

  5. Howard Hughes Medical Institute, Cambridge, MA, USA

    Peyton B. Randolph, Alexander A. Sousa & David R. Liu

  6. BRL Medicine, Inc., Shanghai, China

    Mingyao Liu

  7. School of Biomedical Sciences, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, China

    Bo Feng

Authors
  1. Liang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mengjia Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Changming Luan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hongyi Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gaomeng Ru
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xinyuan Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dujuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shun Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Changwei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jun Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Peyton B. Randolph
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Alexander A. Sousa
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Chao Qu
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Yifan Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Yuting Guan
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Liren Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Mingyao Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Bo Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Gaojie Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. David R. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Dali Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.C., D.R.L. and D.L. designed the experiments. P.R. and A.S. designed prime editing agents. L.C., M.H., C.L., H.G., G.R., X.G., D.Z., S.Z. and C.Q. performed the experiments. L.C., M.H., C.L., H.G., G.R., S.Z., D.Z., J.W., Y.Z, P.R., A.S., C.L., M.L., B.F., G.S., D.R.L. and D.L. analyzed the data. L.C. and D.L. wrote the manuscript, with input from all authors. D.L. supervised the research.

Corresponding author

Correspondence to Dali Li.

Ethics declarations

Competing interests

The authors have submitted patent applications based on the results reported in this study (L.C., D.L., M.H. and C.L.). P.R., A.S. and D.R.L. are co-inventors on prime editing patent applications. D.R.L. is a consultant for Prime Medicine, Beam Therapeutics, Pairwise Plants, Chroma Medicine and Nvelop Therapeutics, companies that use or deliver genome editing or genome engineering agents, and owns equity in these companies. The remaining authors declare no competing interests.

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. 1–15 and Supplementary Note.

Reporting Summary

Supplementary Table

DNA or amino acid sequences of plasmids. List of target sites and primers used in this study. Substrate sequences for inosine excision cleavage assays in vitro. ABE, AXBE and ACBE codon and amino acid conversions.

Supplementary Data

Source data for Supplementary Figs. 2, 3, 5–7 and 9–14

Source data

Source Data Fig. 1

Statistical Source Data for Fig. 1

Source Data Fig. 2

Statistical Source Data for Fig. 2

Source Data Fig. 3

Statistical Source Data for Fig. 3

Source Data Fig. 4

Statistical Source Data for Fig. 4

Source Data Fig. 5

Statistical Source Data for Fig. 5

Source Data Fig. 6

Statistical Source Data for Fig. 6

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

Chen, L., Hong, M., Luan, C. et al. Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos. Nat Biotechnol 42, 638–650 (2024). https://doi.org/10.1038/s41587-023-01821-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-023-01821-9

This article is cited by

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