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Arabidopsis 2010: Targeted mutagenesis in Arabidopsis using zinc finger nucleases

PI: Daniel F. Voytas, University of Minnesota

The next challenge in plant biology is to discern the function of the many genes revealed through the various genome sequencing projects. One of the more conclusive approaches for understanding gene function is to study the phenotypic changes associated with knockout mutations. In Arabidopsis, efficient reverse genetics approaches (e.g. TILLING, transposon and T-DNA mutagenesis) have enabled recovery of knockout mutations in many genes of interest. Functional analysis, however, is often frustrated by the lack of measurable phenotypes due to genetic redundancy. Particularly prevalent in plants are tandem gene duplications, and as many as 15% of Arabidopsis genes are organized in tandem arrays. We propose to develop a targeted mutagenesis protocol to augment existing reverse genetics approaches for understanding Arabidopsis gene function, particularly for genes for which functional analysis has been confounded by genetic redundancy. This technology will also be valuable for plant species such as rice and maize, where genome sequencing projects are ongoing and tandem genes are abundant. Moreover, the ability to generate and regulate specific chromosomal double-strand breaks will facilitate studies of chromosome structure and DNA repair mechanisms in plants.