Abstract

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations.

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Acknowledgements

This work was supported by US National Institutes of Health grants R01 GM088040 (J.K.J. and R.T.P.), R01 GM069906 (J.K.J.), R24 GM078369 (J.K.J.), R01 CA140188 (J.-R.J.Y.), R01 GM081602 (A.J.G.), RC2 MH089956 (A.J.G.), K01 AG031300 (J.-R.J.Y.), K01 AR055619 (D.M.L.) and T32 CA009216 (J.D.S. and J.S.B.); by National Science Foundation grant DBI 0923827 (D.F.V., D.D. and J.K.J.); by the Claflin Distinguished Scholar Award (J.-R.J.Y.); by the Minnesota Soybean Research and Promotion Council (S.J.C. and R.M.S.); and by Alex's Lemonade Stand and the Leukemia Research Foundation (D.M.L.).

Author information

Affiliations

  1. Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Jeffry D Sander
    • , Elizabeth J Dahlborg
    • , Mathew J Goodwin
    • , Jessica S Blackburn
    • , Stacey Thibodeau-Beganny
    • , Morgan L Maeder
    • , Cyd Khayter
    • , David M Langenau
    •  & J Keith Joung
  2. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Jeffry D Sander
    • , Elizabeth J Dahlborg
    • , Mathew J Goodwin
    • , Stacey Thibodeau-Beganny
    • , Morgan L Maeder
    • , Cyd Khayter
    •  & J Keith Joung
  3. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

    • Jeffry D Sander
    • , Jessica S Blackburn
    • , David M Langenau
    •  & J Keith Joung
  4. Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Lindsay Cade
    • , Randall T Peterson
    •  & Jing-Ruey J Yeh
  5. Department of Genetics, Cell Biology and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA.

    • Feng Zhang
    • , Yiping Qi
    • , Christopher J Pierick
    •  & Daniel F Voytas
  6. Genetics Department, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Daniel Cifuentes
    • , Ellen Hoffman
    •  & Antonio J Giraldez
  7. Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota, USA.

    • Shaun J Curtin
    •  & Robert M Stupar
  8. Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA.

    • Morgan L Maeder
    • , David M Langenau
    •  & J Keith Joung
  9. Department of Genetics, Development and Cell Biology and Bioinformatics and Computational Biology Program, Iowa State University, Ames, Iowa, USA.

    • Deepak Reyon
    •  & Drena Dobbs
  10. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

    • Randall T Peterson
    •  & Jing-Ruey J Yeh
  11. Broad Institute, Cambridge, Massachusetts, USA.

    • Randall T Peterson

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Contributions

J.D.S. and J.K.J. conceived of the CoDA engineering method; J.D.S., S.J.C., D.M.L., R.M.S., A.J.G., D.F.V., R.T.P., J.-R.J.Y. and J.K.J. designed research; J.D.S., E.J.D., M.J.G., L.C., F.Z., D.C., S.J.C., J.S.B., S.T.-B., Y.Q., C.J.P., E.H., M.L.M. and C.K. performed experiments; J.D.S., D.R. and D.D. identified potential genomic CoDA target sites; and J.D.S., R.M.S., D.F.V., R.T.P., J.-R.J.Y. and J.K.J. wrote the paper.

Competing interests

F.Z. and D.F.V. are paid for work performed for Cellectis Plant Sciences.

Corresponding author

Correspondence to J Keith Joung.

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DOI

https://doi.org/10.1038/nmeth.1542

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