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Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases

Nature Biotechnology volume 33, pages 179186 (2015) | Download Citation

Abstract

Although great progress has been made in the characterization of the off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification–mediated modification of a previously published high-throughput, genome-wide, translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases more than tenfold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described previously. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.

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Acknowledgements

This work is supported by the National Institutes of Health Grants P01CA109901 and P01AI076210. R.L.F. was supported by the National Institutes of Health NRSA T32CA070083 and is supported by the National Health Institutes of Health NRSA T32AI007512. J.H. is supported by Robertson Foundation/Cancer Research Institute Irvington Fellowship. F.W.A. is an investigator of the Howard Hughes Medical Institute. A549 cells were provided by David Weinstock (Dana-Farber Cancer Institute).

Author information

Author notes

    • Richard L Frock
    •  & Jiazhi Hu

    These authors contributed equally to this work.

Affiliations

  1. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.

    • Richard L Frock
    • , Jiazhi Hu
    • , Robin M Meyers
    • , Yu-Jui Ho
    • , Erina Kii
    •  & Frederick W Alt
  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Richard L Frock
    • , Jiazhi Hu
    • , Robin M Meyers
    • , Yu-Jui Ho
    • , Erina Kii
    •  & Frederick W Alt
  3. Howard Hughes Medical Institute, Boston, Massachusetts, USA.

    • Richard L Frock
    • , Jiazhi Hu
    • , Robin M Meyers
    • , Yu-Jui Ho
    • , Erina Kii
    •  & Frederick W Alt

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Contributions

R.L.F., J.H. and F.W.A. designed the research; R.L.F., J.H. and E.K. performed the research; R.L.F., J.H., R.M.M., Y.-J.H. and E.K. analyzed the data; R.L.F. and F.W.A. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Frederick W Alt.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–16 and Supplementary Tables 1–8

Excel files

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    Supplementary Dataset 1

    Hotspot listing for HTGTS baits

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DOI

https://doi.org/10.1038/nbt.3101