Precise genome modification in the crop species Zea mays using zinc-finger nucleases

Abstract

Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.

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.…”

“…In many cases, ZFN-mediated DSBs have been used to stimulate the HR DNA-repair machinery for HR-mediated gene replacement and gene addition. This approach has been successfully implemented in animals (Beumer et al, 2006;Meng et al, 2008), human cell lines (Urnov et al, 2005;Lombardo et al, 2007), and plants (Wright et al, 2005;Cai et al, 2009;Shukla et al, 2009;Townsend et al, 2009;de Pater et al, 2013). 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.…”

“…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

“…We were able to demonstrate that the integration of a transfer DNA (T-DNA) by homologous recombination (HR) into a specific locus could be enhanced by two orders of magnitude via double-strand-break (DSB) induction using a site-specific endonuclease (8). More recently, by the use of zinc finger nucleases (9), which, in principle, can be used to induce a DSB at any genomic site, endogenous loci have been targeted in Arabidopsis (10), tobacco (11), and maize (12) at high frequencies (13). Some time ago, a method for in vivo targeting was developed in Drosophila.…”

Enhanced gene disruption by programmable nucleases delivered by a minicircle vector

“…The efficiency of homologous recombination is usually more than 10 000 times higher in the presence of engineered nuclease activity. ZFN has been successfully used for targeted genome editing in many systems, such as Drosophila, 7,9 nematode, fish, 10 frog oocytes, 11 rodents, 12 plants 8,[13][14][15] and human cells. [16][17][18][19] TALEN-mediated gene targeting has also been successfully demonstrated in several systems including plants, 20,21 zebrafish, 22 yellow catfish, 23 Caenorhabditis elegans, 24 rats, 25 mice 26 and human cells.…”