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.

  • Brief Communication
  • Published:

High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

Nature Methods volume 8, pages 753–755 (2011)Cite this article

Subjects

Abstract

Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions.

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

Figure 1: ssODN design and genome editing at the human RSK2 locus.
Figure 2: Deletion of chromosomal segments using ssODNs and ZFNs at the human AAVS1 locus.

Similar content being viewed by others

References

  1. Urnov, F.D. et al. Nature 435, 646–651 (2005).

    Article  CAS  Google Scholar 

  2. Moehle, E.A. et al. Proc. Natl. Acad. Sci. USA 104, 3055–3060 (2007).

    Article  CAS  Google Scholar 

  3. Rouet, P., Smih, F. & Jasin, M. Mol. Cell. Biol. 14, 8096–8106 (1994).

    Article  CAS  Google Scholar 

  4. Bibikova, M. et al. Science 300, 764 (2003).

    Article  CAS  Google Scholar 

  5. Porteus, M.H. & Baltimore, D. Science 300, 763 (2003).

    Article  Google Scholar 

  6. Wang, Z. et al. Oligonucleotides 18, 21–32 (2008).

    Article  Google Scholar 

  7. Radecke, S. et al. Mol. Ther. 18, 743–753 (2010).

    Article  CAS  Google Scholar 

  8. Elliott, B. et al. Mol. Cell. Biol. 18, 93–101 (1998).

    Article  CAS  Google Scholar 

  9. Hockemeyer, D. et al. Nat. Biotechnol. 27, 851–857 (2009).

    Article  CAS  Google Scholar 

  10. DeKelver, R.C. et al. Genome Res. 20, 1133–1142 (2010).

    Article  CAS  Google Scholar 

  11. Cohen, M.S. et al. Science 308, 1318–1321 (2005).

    Article  CAS  Google Scholar 

  12. Doehn, U. et al. Mol. Cell 35, 511–522 (2009).

    Article  CAS  Google Scholar 

  13. Santiago, Y. et al. Proc. Natl. Acad. Sci. USA 105, 5809–5814 (2008).

    Article  CAS  Google Scholar 

  14. Lee, H.J., Kim, E. & Kim, J.S. Genome Res. 20, 81–89 (2010).

    Article  CAS  Google Scholar 

  15. Liu, P.Q. et al. Biotechnol. Bioeng. 106, 97–105 (2010).

    CAS  PubMed  Google Scholar 

  16. Storici, F. et al. Proc. Natl. Acad. Sci. USA 100, 14994–14999 (2003).

    Article  CAS  Google Scholar 

  17. Olsen, P.A. et al. DNA Repair (Amst.) 8, 298–308 (2009).

    Article  CAS  Google Scholar 

  18. Aarts, M. & te Riele, H. Nucleic Acids Res. 38, 6956–6967 (2010).

    Article  CAS  Google Scholar 

  19. Soldner, F. et al. Cell published online 14 July 2011 (doi:10.1016/j.cell.2011.06.019).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

M.F. acknowledges support from the Danish Cancer Society (R2-A132-02-S2), the Danish National Research Foundation and the Danish Cancer Research Foundation. J.T. acknowledges support from the US National Institutes of Health (GM071434). We thank our colleagues at Sigma-Aldrich and Sangamo Biosciences for helpful advice and discussion; H. Holemon, P. Sullivan and D. Smoller for support; and D. Carroll for helpful comments and discussion.

Author information

Authors and Affiliations

  1. Sigma-Aldrich Biotechnology, St. Louis, Missouri, USA

    Fuqiang Chen, Shondra M Pruett-Miller, Yuping Huang, Monika Gjoka, Trevor N Collingwood & Gregory D Davis

  2. Biotech Research and Innovation Centre and Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark

    Katarzyna Duda & Morten Frodin

  3. Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, USA

    Jack Taunton

Authors
  1. Fuqiang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shondra M Pruett-Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuping Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Monika Gjoka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Katarzyna Duda
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jack Taunton
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Trevor N Collingwood
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Morten Frodin
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gregory D Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.C., S.M.P.-M., Y.H., M.G. and K.D. performed experiments. G.D.D., F.C., S.M.P.-M. and M.F. designed experiments. G.D.D., T.N.C., M.F., F.C., S.M.P.-M. and J.T. wrote the paper.

Corresponding author

Correspondence to Gregory D Davis.

Ethics declarations

Competing interests

F.C., S.M.P.-M., Y.H., M.G., T.N.C. and G.D.D. are employees of Sigma-Aldrich Corp.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18, Supplementary Tables 1–2, Supplementary Notes 1–3 (PDF 1402 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, F., Pruett-Miller, S., Huang, Y. et al. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat Methods 8, 753–755 (2011). https://doi.org/10.1038/nmeth.1653

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1653

This article is cited by

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