Validation and expression of zinc finger nucleases in plant cells

Zinc finger nucleases (ZFNs) can be designed to target virtually any long stretch of DNA sequence. Their expression in living cells has been shown to lead to gene targeting via homologous recombination, site-specific mutagenesis, and targeted DNA integration in various species. A variety of assays have been developed to test ZFN activity both in vitro and in vivo, and an assortment of vectors have been constructed to facilitate the analysis and expression of ZFNs in mammalian, and specifically human cells, as well as in other model organisms. Here we describe a set of protocols and vectors that were specifically designed to analyze ZFN activity in plant cells. Our assays provide the user with versatile tools and simple protocols for in-planta analysis of ZFN activity on transiently delivered and stably integrated mutated plant reporter (GUS)-encoding genes. Specifically designed for maximum compatibility with a generalized plant expression system, our vector system also allows easy assembly of ZFN plant transformation vectors for gene-targeting experiments in plants.