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A Rapid and General Assay for Monitoring Endogenous Gene Modification

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Engineered Zinc Finger Proteins

Abstract

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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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

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