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:

An adenine base editor variant expands context compatibility

Nature Biotechnology (2024)Cite this article

Subjects

Abstract

Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. Existing ABEs function most effectively when the target A is in a TA context. Here we evolve the Escherichia coli transfer RNA-specific adenosine deaminase (TadA) to generate TadA8r, which extends potent deoxyadenosine deamination to RA (R = A or G) and is faster in processing GA than TadA8.20 and TadA8e, the two most active TadA variants reported so far. ABE8r, comprising TadA8r and a Streptococcus pyogenes Cas9 nickase, expands the editing window at the protospacer adjacent motif-distal end and outperforms ABE7.10, ABE8.20 and ABE8e in correcting disease-associated G:C-to-A:T transitions in the human genome, with a controlled off-target profile. We show ABE8r-mediated editing of clinically relevant sites that are poorly accessed by existing editors, including sites in PCSK9, whose disruption reduces low-density lipoprotein cholesterol, and ABCA4-p.Gly1961Glu, the most frequent mutation in Stargardt disease.

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: Genotypes of WT TadA and TadA variants.
Fig. 2: Directed evolution of TadA for RA compatibility.
Fig. 3: Characterization of ABE8r in HEK293T cells and in vitro.
Fig. 4: Off-target assessment for ABE8r.
Fig. 5: Evaluation of ABEs using a paired sgRNA-target library.
Fig. 6: ABE8r-mediated A:T-to-G:C editing at disease-associated loci.

Similar content being viewed by others

Data availability

All next-generation sequencing data have been deposited to the NCBI’s Gene Expression Omnibus and can be accessed through accession no. GSE243181 (ref. 65). Amplicon sequencing data have been deposited to the NCBI Sequence Read Archive under BioProject no. PRJNA925224.

References

  1. Landrum, M. J. et al. ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 42, D980–D985 (2014).

    Article  CAS  PubMed  Google Scholar 

  2. 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 

  3. 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  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. 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 

  5. Zeng, Y. et al. Correction of the Marfan syndrome pathogenic FBN1 mutation by base editing in human cells and heterozygous embryos. Mol. Ther. 26, 2631–2637 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ryu, S. M. et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat. Biotechnol. 36, 536–539 (2018).

    Article  CAS  PubMed  Google Scholar 

  7. Liu, Z. et al. Highly efficient RNA-guided base editing in rabbit. Nat. Commun. 9, 2717 (2018).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  8. Song, C. Q. et al. Adenine base editing in an adult mouse model of tyrosinaemia. Nat. Biomed. Eng. 4, 125–130 (2020).

    Article  CAS  PubMed  Google Scholar 

  9. Li, C. et al. Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion. Genome Biol. 19, 59 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Hua, K., Tao, X., Yuan, F., Wang, D. & Zhu, J. K. Precise A•T to G•C base editing in the rice genome. Mol. Plant 11, 627–630 (2018).

    Article  CAS  PubMed  Google Scholar 

  11. Yan, F. et al. Highly efficient A.T to G.C base editing by Cas9n-guided tRNA adenosine deaminase in rice. Mol. Plant 11, 631–634 (2018).

    Article  CAS  PubMed  Google Scholar 

  12. Koblan, L. W. et al. In vivo base editing rescues Hutchinson–Gilford progeria syndrome in mice. Nature 589, 608–614 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  13. Newby, G. A. et al. Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature 595, 295–302 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Arbab, M. et al. Base editing rescue of spinal muscular atrophy in cells and in mice. Science 380, eadg6518 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Musunuru, K. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429–434 (2021).

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Rothgangl, T. et al. In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels. Nat. Biotechnol. 39, 949–957 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhang, W. et al. Multiplex precise base editing in cynomolgus monkeys. Nat. Commun. 11, 2325 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lee, R. G. et al. Efficacy and safety of an investigational single-course CRISPR base editing therapy targeting PCSK9 in non-human primate and mouse models. Circulation 147, 242–253 (2023).

    Article  CAS  PubMed  Google Scholar 

  19. Wolf, J., Gerber, A. P. & Keller, W. TadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. EMBO J. 21, 3841–3851 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 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 

  22. Song, M. et al. Sequence-specific prediction of the efficiencies of adenine and cytosine base editors. Nat. Biotechnol. 38, 1037–1043 (2020).

    Article  CAS  PubMed  Google Scholar 

  23. Kim, N. et al. Deep learning models to predict the editing efficiencies and outcomes of diverse base editors. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01792-x (2023).

  24. Gaudelli, N. M. et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat. Biotechnol. 38, 892–900 (2020).

    Article  CAS  PubMed  Google Scholar 

  25. 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 

  26. Li, J. et al. Structure-guided engineering of adenine base editor with minimized RNA off-targeting activity. Nat. Commun. 12, 2287 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Fang, G. et al. Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing. Nat. Biotechnol. 30, 1232–1239 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Marinus, M. G. & Lobner-Olesen, A. DNA methylation. EcoSal Plus https://doi.org/10.1128/ecosalplus.ESP-0003-2013 (2014).

  29. Losey, H. C., Ruthenburg, A. J. & Verdine, G. L. Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA. Nat. Struct. Mol. Biol. 13, 153–159 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Cadwell, R. C. & Joyce, G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2, 28–33 (1992).

    Article  CAS  PubMed  Google Scholar 

  31. Grunewald, J. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat. Biotechnol. 37, 1041–1048 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Thuronyi, B. W. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol. 37, 1070–1079 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471–481 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 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  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wang, X. et al. Cas12a base editors induce efficient and specific editing with low DNA damage response. Cell Rep. 31, 107723 (2020).

    Article  CAS  PubMed  Google Scholar 

  38. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kleinstiver, B. P. et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat. Biotechnol. 33, 1293–1298 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kleinstiver, B. P. et al. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    Article  CAS  PubMed  Google Scholar 

  43. Liang, P. et al. Genome-wide profiling of adenine base editor specificity by EndoV-seq. Nat. Commun. 10, 67 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  44. Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  45. Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  46. 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 

  47. Yu, Y. et al. Cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity. Nat. Commun. 11, 2052 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  48. Wang, L. et al. Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations. Nat. Cell Biol. 23, 552–563 (2021).

    Article  PubMed  Google Scholar 

  49. Jin, S. et al. Rationally designed APOBEC3B cytosine base editors with improved specificity. Mol. Cell 79, 728–740 e726 (2020).

    Article  CAS  PubMed  Google Scholar 

  50. Rees, H. A., Wilson, C., Doman, J. L. & Liu, D. R. Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci. Adv. 5, eaax5717 (2019).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  51. Grunewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  52. 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 

  53. Sanchez-Rivera, F. J. et al. Base editing sensor libraries for high-throughput engineering and functional analysis of cancer-associated single nucleotide variants. Nat. Biotechnol. 40, 862–873 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Rivera, A. et al. A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration. Am. J. Hum. Genet. 67, 800–813 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Fujinami, K. et al. Detailed genetic characteristics of an international large cohort of patients with Stargardt disease: ProgStar study report 8. Br. J. Ophthalmol. 103, 390–397 (2019).

    Article  PubMed  Google Scholar 

  56. Muller, A. et al. High-efficiency base editing for Stargardt disease in mice, non-human primates, and human retina tissue. Preprint at bioRxiv https://doi.org/10.1101/2023.04.17.535579 (2023).

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

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  58. Xiao, Y. L. et al. Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination. Nat. Biotechnol. 41, 993–1003 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Lapinaite, A. et al. DNA capture by a CRISPR-Cas9-guided adenine base editor. Science 369, 566–571 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  60. Ranzau, B. L., Rallapalli, K. L., Evanoff, M., Paesani, F. & Komor, A. C. The wild-type tRNA adenosine deaminase enzyme TadA is capable of sequence-specific DNA base editing. ChemBioChem 24, e202200788 (2023).

    Article  CAS  PubMed  Google Scholar 

  61. Kohli, R. M. et al. A portable hot spot recognition loop transfers sequence preferences from APOBEC family members to activation-induced cytidine deaminase. J. Biol. Chem. 284, 22898–22904 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Carpenter, M. A., Rajagurubandara, E., Wijesinghe, P. & Bhagwat, A. S. Determinants of sequence-specificity within human AID and APOBEC3G. DNA Repair 9, 579–587 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Wang, M., Rada, C. & Neuberger, M. S. Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID. J. Exp. Med. 207, 141–153 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. 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 

  65. Xiao, Y. L., Wu, Y. & Tang, W. Directed evolution of an adenine base editor with increased activity and context compatibility. Gene Expression Omnibus https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE243181 (2023).

Download references

Acknowledgements

We thank H. Yan for optimizing the transfection workflow and K. M. Watters for scientific editing of the paper. This work was completed in part with computing resources provided by the University of Chicago Research Computing Center. We thank the Single Cell Immunophenotyping Core Facility at the University of Chicago for sequencing support. W.T. is supported by the Searle Scholars Program (grant no. SSP-2021-113), the Cancer Research Foundation Young Investigator Program, the American Cancer Society (grant no. RSG-22-043-01-ET) and the David & Lucile Packard Foundation (grant no. 2022-74685).

Author information

Authors and Affiliations

  1. Department of Chemistry, The University of Chicago, Chicago, IL, USA

    Yu-Lan Xiao, Yuan Wu & Weixin Tang

  2. Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Yu-Lan Xiao, Yuan Wu & Weixin Tang

Authors
  1. Yu-Lan Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuan Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Weixin Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.-L.X. and W.T. conceived and designed the study. Y.-L.X. carried out directed evolution, purified and characterized TadA8r, constructed the paired sgRNA-target library and evaluated TadA8r in human cells. Y.W. assisted with deaminase purification and characterization. Y.W. analyzed transcriptome-wide off-target effects for all ABEs and editing data generated using the paired sgRNA-target library. W.T. supervised the study. Y.-L.X., Y.W. and W.T. wrote the paper.

Corresponding author

Correspondence to Weixin Tang.

Ethics declarations

Competing interests

A patent has been filed for TadA8r and its applications in gene editing by the University of Chicago.

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 Notes 1–4, Tables 1–10 and Figs. 1–39.

Reporting Summary

Supplementary Table 1

Evaluated ABE targets of clinical relevance_full table.

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

Xiao, YL., Wu, Y. & Tang, W. An adenine base editor variant expands context compatibility. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-023-01994-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41587-023-01994-3

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing