High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases

Abstract

We report here an efficient method for targeted mutagenesis of Arabidopsis genes through regulated expression of zinc finger nucleases (ZFNs)-enzymes engineered to create DNA doublestrand breaks at specific target loci. ZFNs recognizing the Arabidopsis ADH1 and TT4 genes were made by Oligomerized Pool ENgineering (OPEN)-a publicly available, selection-based platform that yields high quality zinc finger arrays. The ADH1 and TT4 ZFNs were placed under control of an estrogen-inducible promoter and introduced into Arabidopsis plants by floral-dip transformation. Primary transgenic Arabidopsis seedlings induced to express the ADH1 or TT4 ZFNs exhibited somatic mutation frequencies of 7% or 16%, respectively. The induced mutations were typically insertions or deletions (1-142 bp) that were localized at the ZFN cleavage site and likely derived from imprecise repair of chromosome breaks by nonhomologous end-joining. Mutations were transmitted to the next generation for 69% of primary transgenics expressing the ADH1 ZFNs and 33% of transgenics expressing the TT4 ZFNs. Furthermore, ≈20% of the mutant-producing plants were homozygous for mutations at ADH1 or TT4, indicating that both alleles were disrupted. ADH1 and TT4 were chosen as targets for this study because of their selectable or screenable phenotypes (adh1, allyl alcohol resistance; tt4, lack of anthocyanins in the seed coat). However, the high frequency of observed ZFNinduced mutagenesis suggests that targeted mutations can readily be recovered by simply screening progeny of primary transgenic plants by PCR and DNA sequencing. Taken together, our results suggest that it should now be possible to obtain mutations in any Arabidopsis target gene regardless of its mutant phenotype.nonhomologous end-joining | gene knockout | alcohol dehydrogenase | chalcone synthase

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

“…In previous studies, HR-mediated gene-replacement strategies have been the preferred mode of gene replacement in plants (Wright et al, 2005;Shukla et al, 2009;Townsend et al, 2009;de Pater et al, 2013), while NHEJ-mediated repair of ZFN-induced DSBs has been limited to targeted mutagenesis and transgene removal (Lloyd et al, 2005;Morton et al, 2006;Maeder et al, 2008;de Pater et al, 2009;Tovkach et al, 2009;Osakabe et al, 2010;Petolino et al, 2010;Zhang et al, 2010). Our findings show that the NHEJ DNA-repair pathway can be harnessed for gene replacement in plant species.…”

“…However, it is important to note that while ZFNinduced DSBs can indeed induce the HR repair machinery, most of these breaks are repaired by the cell's NHEJ DNA-repair machinery in plant and other species. Thus, ZFNs have also been used for the induction of site-specific mutagenesis in many eukaryotic cells, including plant cells (Lloyd et al, 2005;Tovkach et al, 2009;Marton et al, 2010;Osakabe et al, 2010;Zhang et al, 2010;Curtin et al, 2011;Even-Faitelson et al, 2011). More recently, ZFNs have been used for NHEJmediated targeted chromosomal deletions (Lee et al, 2010;Söllü et al, 2010), transgene removal (Petolino et al, 2010), and even NHEJ-mediated targeted DNA integration in mammalian genomes (Orlando et al, 2010).…”

“…ZFNs are artificial hybrid restriction enzymes that are composed of a fusion between a designed zinc-finger protein DNA-binding domain and the cleavage domain of the FokI endonuclease (for review, see Porteus, 2009;Urnov et al, 2010;Weinthal et al, 2010;Tzfira et al, 2012). ZFNs have been designed to target a wide variety of native and artificial sequences in human, animal, and plant cells (Kumar et al, 2006;Porteus, 2006;Lombardo et al, 2007;Moehle et al, 2007;Doyon et al, 2008;Cai et al, 2009;Shukla et al, 2009;Liu et al, 2010;Zhang et al, 2010;de Pater et al, 2013). In many cases, ZFN-mediated DSBs have been used to stimulate the HR DNA-repair machinery for HR-mediated gene replacement and gene addition.…”

In planta gene targeting

“…The most obvious application of such an approach is the use of artificially constructed modular endonucleases, such as meganucleases (26), zinc finger nucleases (27), or transcription activator-like effector nucleases (28), that are designed to cut at a specific site in the gene of interest. Recent studies demonstrated that the aforementioned nucleases can be applied in the same way as I-SceI used in our study for different DSB-induced recombination reactions in plants (8,15,19,29).…”

Discover and Connect Cellular Signaling

“…It is also important to be able to obtain targeted mutations and to design new mutants using zinc finger nucleases and novel means in plants for functional characterization ( Fig. 2; Zhang et al, 2010).…”