Targeted transgene integration in plant cells using designed zinc finger nucleases

Abstract

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

“…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 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.…”

Pollen Sterility—A Promising Approach to Gene Confinement and Breeding for Genetically Modified Bioenergy Crops

“…Analysis of expression characteristics of a maize pollen specific promoter (Zm13Pro) shows that when linked to a reporter gene, such as gusA, a subset of transgenics is pollen specific and when linked to a cytotoxic gene (i.e., Zm13Pro::barnase) Zm13Pro can confer pollen sterility. Perhaps stable knockouts could be created against orthologous targets using Zinc Finger Nuclease (ZFN) or Transcription Activator-Like Effector Nuclease (TALEN) modifications to direct sterility functions [62][63][64][65][66][67][68][69] avoiding any potential side effects of low levels of cytotoxin gene expression in non-target tissues. This study also shows that pollen specific expression is co-integrated and capable of co-expression when linked to a trait gene of interest.…”

“…These T 0 plantlets show PCR products consistent with the expected 494 bp ( Figure 3). Verification of the presence of the hpt transgene was accomplished via hpt-specific primers using the identical controls; the same T 0 plants (31,69,227,344,625,680,717, and 718, respectively) tested positive for the presence of the hpt transgene as these T 0 events yielded PCR products consistent with the expected 362 bp hpt product ( Figure 4). All 44 T 0 plants which had been regenerated from hygromycin resistant calli tested positive for the gusA and hpt transgenes, indicating that each regenerated transgenic event carried at least one intact copy of both the gusA and hpt transgene cassettes.…”

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

“…One of the most promising application of these agents is gene therapy [1][2][3][4][5], but they are also used for gene modifications in cells to create animal models [6,7], to improve properties of plants (e.g. targeted addition of a herbicide-tolerance gene [8]) [9,10], and to broaden our knowledge on genetic phenomena [11]. The basis of these experiments is that the double-strand cleavage of DNA induces the cells' repair machinery to correct the damaged molecule.…”