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Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases

Nature Biotechnology volume 43, pages 923–935 (2025)Cite this article

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Abstract

Genome editing technologies based on DNA-dependent polymerases (DDPs) could offer several benefits compared with other types of editors to install diverse edits. Here, we develop click editing, a genome writing platform that couples the advantageous properties of DDPs with RNA-programmable nickases to permit the installation of a range of edits, including substitutions, insertions and deletions. Click editors (CEs) leverage the ‘click’-like bioconjugation ability of HUH endonucleases with single-stranded DNA substrates to covalently tether ‘click DNA’ (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs and their clkDNAs, we demonstrate the ability to install precise genome edits with minimal indels in diverse immortalized human cell types and primary fibroblasts with precise editing efficiencies of up to ~30%. Editing efficiency can be improved by rapidly screening clkDNA oligonucleotides with various modifications, including repair-evading substitutions. Click editing is a precise and versatile genome editing approach for diverse biological applications.

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Fig. 1: Overview and development of click editing.
Fig. 2: Optimization of clkDNA parameters.
Fig. 3: DNA repair evasion through clkDNA modification.
Fig. 4: Comparison to prime editing and off-target analyses.
Fig. 5: Architectural variations and click editing efficiency in different human cellular models and mRNA delivery.

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Data availability

Primary datasets are available in the Source data and Supplementary Table 10. Sequencing datasets are available from the NCBI Sequence Read Archive (SRA) under BioProject ID PRJNA1015647 (ref. 112). Source data are provided with this paper.

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Acknowledgements

We acknowledge members of the Kleinstiver laboratory for critical feedback, A. Anzalone for advice regarding data analysis, E. J. Sontheimer and W. Xue for communicating results before publication and providing the mRNA expression plasmid, L. Pinello for advice regarding analysis of clkDNA parameter trends, J. Ruliera from the Mass General Brigham (MGB) Tissue Culture Core Services for deriving fibroblasts, M. N. Whittaker for cloning the pegRNA entry vector and Z. Hebert and M. Berkeley from the Dana–Farber Cancer Institute Molecular Biology Core Facilities for sequencing support. We acknowledge support from an EMBO Long Term Fellowship (ALTF 750-2022; to J.F.d.S.), a National Science Foundation Graduate Research Fellowship (2020295403; C.J.T.), a Massachusetts General Hospital (MGH) Executive Committee on Research Fund for Medical Discovery Fundamental Research Fellowship Award (L.M.), a Swiss National Science Foundation grant (P180777; F.M.C.B.), a Friedreich’s Ataxia Research Alliance (FARA) and FARA Australia Fellowship (D.R.R.), a Banting (Natural Sciences and Engineering Research Council of Canada) Postdoctoral Fellowship (C.R.C), an MGH Howard M. Goodman Fellowship (B.P.K), the Kayden–Lambert MGH Research Scholar Award 2023–2028 (B.P.K) and National Institutes of Health grants R35GM142553 (L.H.C.), U54NS115052 (F.S.E.), UM1HG012010 (B.P.K.), P01HL142494 (B.P.K.) and DP2CA281401 (B.P.K.).

Author information

Author notes

  1. These authors contributed equally: Joana Ferreira da Silva, Connor J. Tou.

Authors and Affiliations

  1. Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA

    Joana Ferreira da Silva, Connor J. Tou, Emily M. King, Madeline L. Eller, David Rufino-Ramos, Linyuan Ma, Christopher R. Cromwell, Jasna Metovic, Florian S. Eichler & Benjamin P. Kleinstiver

  2. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA

    Joana Ferreira da Silva, Connor J. Tou, Emily M. King, Madeline L. Eller, David Rufino-Ramos, Linyuan Ma, Christopher R. Cromwell & Benjamin P. Kleinstiver

  3. Department of Pathology, Harvard Medical School, Boston, MA, USA

    Joana Ferreira da Silva, David Rufino-Ramos, Linyuan Ma, Christopher R. Cromwell & Benjamin P. Kleinstiver

  4. Biological Engineering Program, Massachusetts Institute of Technology, Cambridge, MA, USA

    Connor J. Tou

  5. Biological and Biomedical Sciences Program, Harvard University, Boston, MA, USA

    Emily M. King

  6. Department of Neurology, Massachusetts General Hospital, Boston, MA, USA

    Jasna Metovic & Florian S. Eichler

  7. Department of Neurology, Harvard Medical School, Boston, MA, USA

    Jasna Metovic & Florian S. Eichler

  8. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA

    Friederike M. C. Benning & Luke H. Chao

  9. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Friederike M. C. Benning & Luke H. Chao

Authors
  1. Joana Ferreira da Silva
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  2. Connor J. Tou
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  3. Emily M. King
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  4. Madeline L. Eller
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  5. David Rufino-Ramos
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  6. Linyuan Ma
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  7. Christopher R. Cromwell
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  8. Jasna Metovic
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  9. Friederike M. C. Benning
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  10. Luke H. Chao
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  11. Florian S. Eichler
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  12. Benjamin P. Kleinstiver
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Contributions

C.J.T. conceived of the initial concept with subsequent ideation by J.F.d.S., C.J.T., B.P.K. and other co-authors. J.F.d.S., C.J.T., M.L.E., E.M.K., L.M., D.R.R., C.R.C. and J.M. designed and performed experiments. F.M.C.B. and L.H.C. contributed to biochemical approaches. B.P.K. contributed to the experimental design and oversaw the study. J.F.d.S., C.J.T. and B.P.K. wrote the manuscript with contributions and/or revisions from all authors.

Corresponding author

Correspondence to Benjamin P. Kleinstiver.

Ethics declarations

Competing interests

J.F.d.S., C.J.T. and B.P.K. are inventors on a patent application filed by the MGB that describes click editing. C.J.T. and B.P.K. are inventors on additional patents or patent applications filed by the MGB that describe genome engineering technologies. F.S.E. has consulted for Sanofi, Spur Therapeutics, Taysha, ASPA Therapeutics and Atlas Venture. B.P.K. is a consultant for EcoR1 Capital and Novartis Venture Fund and is on the scientific advisory board of Acrigen Biosciences, Life Edit Therapeutics and Prime Medicine. B.P.K. has a financial interest in Prime Medicine, a company developing therapeutic CRISPR–Cas technologies for gene editing. B.P.K.’s interests were reviewed and are managed by the MGH and MGB in accordance with their conflict-of-interest policies. The other authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Notes 1–4, Supplementary Figs. 1–25 and Supplementary References.

Reporting Summary

Supplementary Tables 1–9

Supplementary Tables 1–9 separated by tabs of the same file.

Supplementary Table 10

Source data from the supplementary figures.

Source data

Source Data Figures 1–5

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Ferreira da Silva, J., Tou, C.J., King, E.M. et al. Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases. Nat Biotechnol 43, 923–935 (2025). https://doi.org/10.1038/s41587-024-02324-x

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