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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