Abstract

Zinc-finger nucleases (ZFNs) allow gene editing in live cells by inducing a targeted DNA double-strand break (DSB) at a specific genomic locus. However, strategies for characterizing the genome-wide specificity of ZFNs remain limited. We show that nonhomologous end-joining captures integrase-defective lentiviral vectors at DSBs, tagging these transient events. Genome-wide integration site analysis mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or IL2RG. Ranking loci with repeatedly detectable nuclease activity by deep-sequencing allowed us to monitor the degree of ZFN specificity in vivo at these positions. Cleavage required binding of ZFNs in specific spatial arrangements on DNA bearing high homology to the intended target site and only tolerated mismatches at individual positions of the ZFN binding sites. Whereas the consensus binding sequence derived in vivo closely matched that obtained in biochemical experiments, the ranking of in vivo cleavage sites could not be predicted in silico. Comprehensive mapping of ZFN activity in vivo will facilitate the broad application of these reagents in translational research.

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Acknowledgements

We thank U. Abel for fruitful discussions. Funding was provided by the Deutsche Forschungsgemeinschaft (SPP1230, grant of the Tumor Center Heidelberg/Mannheim), by the Bundesministerium für Bildung und Forschung (iGene), by the VIth + VIIth Framework Programs of the European Commission (EC, European Network for the Advancement of Clinical Gene Transfer and Therapy (CLINIGENE) and Persisting Transgenesis (PERSIST) and by the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Immunotherapy of Cancer to C.v.K. and M.S. Funding to L.N. was provided by Telethon (TIGET grant), EC (FP7-HEALTH-2009-222878, PERSIST; ERC Advanced Grant FP7, Targeting gene therapy - 249845).

Author information

Author notes

    • Richard Gabriel
    • , Angelo Lombardo
    • , Manfred Schmidt
    • , Luigi Naldini
    •  & Christof von Kalle

    These authors contributed equally to this work.

Affiliations

  1. Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.

    • Richard Gabriel
    • , Anne Arens
    • , Christine Kaeppel
    • , Ali Nowrouzi
    • , Cynthia C Bartholomae
    • , Hanno Glimm
    • , Manfred Schmidt
    •  & Christof von Kalle
  2. San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Vita Salute San Raffaele University, Milan, Italy.

    • Angelo Lombardo
    • , Pietro Genovese
    •  & Luigi Naldini
  3. Sangamo BioSciences, Inc., Richmond, California, USA.

    • Jeffrey C Miller
    • , Jianbin Wang
    • , Geoffrey Friedman
    • , Michael C Holmes
    •  & Philip D Gregory

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Contributions

R.G., A.L., H.G., M.S., J.C.M., P.D.G., M.C.H., L.N. and C.v.K. conceived the project, designed experiments and interpreted data. R.G., A.L., P.G., C.K., A.N., J.W., G.F. and C.C.B. performed experiments. R.G., A.A. and J.C.M. conducted bioinformatics analysis. M.C.H. and P.D.G. provided ZFN. R.G., A.L., A.A., J.C.M., M.C.H., P.D.G., M.S., L.N. and C.v.K. prepared and wrote the manuscript.

Competing interests

J.C.M., J.W., G.F., M.C.H. and P.D.G. are full-time employees of Sangamo BioSciences.

Corresponding authors

Correspondence to Luigi Naldini or Christof von Kalle.

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    Supplementary Tables 1,2, Supplementary Discussion, Supplementary Methods and Supplementary Figures 1–16

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DOI

https://doi.org/10.1038/nbt.1948

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