In vitro assessment of zinc finger nuclease activity

Methods Mol Biol. 2010:649:227-35. doi: 10.1007/978-1-60761-753-2_13.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell-Free System / metabolism
  • DNA Breaks, Double-Stranded
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Humans
  • Zinc Fingers / genetics*

Substances

  • Endonucleases