Gene targeting in maize by somatic ectopic recombination

Abstract

SummaryLow transformation efficiency and high background of non-targeted events are major constraints to gene targeting in plants. We demonstrate here applicability in maize of a system that reduces the constraint from transformation efficiency. The system requires regenerable transformants in which all of the following elements are stably integrated in the genome: (i) donor DNA with the gene of interest adjacent to sequence for repair of a defective selectable marker, (ii) sequence encoding a rare-cutting endonuclease such as I-SceI, (iii) a target locus (TL) comprising the defective selectable marker and I-SceI cleavage site. Typically, this requires additional markers for the integration of the donor and target sequences, which may be assembled through cross-pollination of separate transformants. Inducible expression of I-SceI then cleaves the TL and facilitates homologous recombination, which is assayed by selection for the repaired marker. We used bar and gfp markers to identify assembled transformants, a dexamethasone-inducible I-SceI::GR protein, and selection for recombination events that restored an intact nptII. Applying this strategy to callus permitted the selection of recombination into the TL at a frequency of 0.085% per extracted immature embryo (29% of recombinants). Our results also indicate that excision of the donor locus (DL) through the use of flanking I-SceI cleavage sites may be unnecessary, and a source of unwanted repair events at the DL. The system allows production, from each assembled transformant, of many cells that subsequently can be treated to induce gene targeting. This may facilitate gene targeting in plant species for which transformation efficiencies are otherwise limiting.

A modular gene targeting system for sequential transgene stacking in plants

“…Most of the crop plants and cultivars, however, are more recalcitrant for transformation; selection would therefore be a key for the routine application of gene targeting technology in plants. Selection-mediated gene targeting systems have been successfully demonstrated previously using a non-functional selection (Ayar et al, 2013;Cai et al, 2009;Puchta et al, 1996) or reporter (Fauser et al, 2012) genes in the target locus. The targeted insertion of the donor construct into the target locus complements the non-functional selectable marker, providing desired positive selection for gene targeting.…”

Gene targeting in plants: 25 years later

“…A first indication that this may be possible comes from a recent study in maize with a similar system that used marker genes and the endonuclease I-SceI. The authors demonstrated that also an ectopic copy of the targeting vector could be used as a template after DSB induction at the target locus (Ayar et al, 2012).…”

DNA Replicons for Plant Genome Engineering

“…Protoplasts can be transformed at high efficiency, but only a handful of plants can be effectively regenerated from protoplasts. Alternatively, constructs encoding nucleases and repair templates can be integrated into plant genomes (Fauser et al, 2012;Ayar et al, 2013). This so-called in planta genome engineering strategy relies on the gene targeting to occur during the life of the plant, with the anticipation that germline cells will be modified and ultimately give rise to seed with the desired sequence alterations.…”