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A Rapid and General Assay for Monitoring Endogenous Gene Modification

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Engineered Zinc Finger Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 649))

Abstract

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The development of zinc finger nucleases for targeted gene modification can benefit from rapid functional assays that directly quantify activity at the endogenous target. Here we describe a simple procedure for quantifying mutations that result from DNA double-strand break repair via non-homologous end joining. The assay is based on the ability of the Surveyor nuclease to selectively cleave distorted duplex DNA formed via cross-annealing of mutated and wild-type sequence.

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Acknowledgments

We thank Elo Leung, Xiangdong Meng, Sarah Hinkley, and Lei Zhang for help with the design and assembly of ZFNs; Jianbin Wang and Geoff Friedman for transfections; and Philip Gregory, Susan Abrahamson, and Lei Zhang for helpful comments on the manuscript.

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Authors and Affiliations

  1. Sangamo BioSciences, Inc., Richmond, CA, USA

    Dmitry Y. Guschin, Adam J. Waite, George E. Katibah, Jeffrey C. Miller, Michael C. Holmes & Edward J. Rebar

  2. Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Adam J. Waite

Authors
  1. Dmitry Y. Guschin
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  2. Adam J. Waite
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  3. George E. Katibah
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  4. Jeffrey C. Miller
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  5. Michael C. Holmes
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  6. Edward J. Rebar
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Editor information

Editors and Affiliations

  1. School of Molecular &, Microbial Biosciences, University of Sydney, Cnr. Butlin Ave. & Maze Cres., Sydney, 2006, New South Wales, Australia

    Joel P. Mackay

  2. Dept. Pharmacology & Genome Center, University of California, Davis, E. Health Sciences Drive 451, Davis, 95616, California, USA

    David J. Segal

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Guschin, D.Y., Waite, A.J., Katibah, G.E., Miller, J.C., Holmes, M.C., Rebar, E.J. (2010). A Rapid and General Assay for Monitoring Endogenous Gene Modification. In: Mackay, J., Segal, D. (eds) Engineered Zinc Finger Proteins. Methods in Molecular Biology, vol 649. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-753-2_15

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  • DOI: https://doi.org/10.1007/978-1-60761-753-2_15

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-752-5

  • Online ISBN: 978-1-60761-753-2

  • eBook Packages: Springer Protocols

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