In vitro assessment of zinc finger nuclease activity

Abstract

The technical advances in developing artificial endonucleases, such as zinc finger nucleases (ZFNs), have opened a wide field of applications in the genome engineering arena, including the therapeutic correction of mutated genes in the human genome. Gene editing frequencies of up to 50% in human cells under non-selective conditions reveal the power of the ZFN technology. Activity and toxicity of ZFNs are determined by a number of parameters, including the specificity of DNA binding, the kinetics of dimerization of the two ZFN subunits, and the catalytic activity. In order to investigate these parameters individually, a cell-free system that models these reactions is essential. Here, we present a simple and fast method for the functional testing of ZFNs in vitro. © 2010 Springer Science+Business Media, LLC.