A Rapid and General Assay for Monitoring Endogenous Gene Modification
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.
Key wordsZinc finger nuclease (ZFN) designed zinc finger proteins non-homologous end joining (NHEJ) Surveyor nuclease Cel 1 genome modification
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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