Targeted Mutagenesis in Arabidopsis Using Zinc-Finger Nucleases

Abstract

We report here an efficient method for making targeted mutations in Arabidopsis thaliana genes. The approach uses zinc-finger nucleases (ZFNs) – enzymes engineered to create DNA double-strand breaks at specific target loci. Imprecise repair of double-strand breaks by nonhomologous end-joining generates small insertions or deletions at the cleavage site. In this protocol, constructs encoding ZFNs for specific loci are transformed into Arabidopsis by Agrobacterium-mediated transformation. ZFN expression is induced during germination to initiate mutagenesis of the target locus. Typically, more than 20% of the primary transgenics segregate loss-of-function mutations in the next generation. ZFNs make it possible to expand the range of Arabidopsis mutants available for study and to create mutations in genes missed by random mutagenesis approaches, such as those using T-DNA, transposons, or chemical mutagens.