Trait stacking via targeted genome editing

Abstract

SummaryModern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high-efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular 'trait landing pads' (TLPs) that allow 'mix-and-match', on-demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease-driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS TM -mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo-derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease-driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFNmediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking.

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline

“…Thus, it is possible to perform specific modifications to a DNA sequence, such as gene insertions, replacements or deletions [including transgenes; Chen and Gao (2013), Rinaldo and Ayliffe (2015)]. Moreover, when using TALENs or CRISPR/Cas9, it is possible to perform gene stacking to introduce multiple genes in one locus; this increases the stability and facilitates inheritance of thus obtained traits (Ainley et al, 2013;D'Halluin et al, 2013). If using multiple nucleases at once, it is also possible to perform large DNA deletions (allowing removal of entire gene regulatory networks), inversions, or translocations (Chen and Gao, 2013).…”

“…The whole stack of the four transgenes was inherited in the subsequent generations as a single genetic unit and field experiments confirmed that all of the genes were expressed. Ainley et al (2013) introduced pat and aad1 genes that confer resistance to glufosinate and aryloxyphenoxypropionate herbicides, respectively, in maize. The gene knock-in was performed in two steps using ZFNs.…”

Vast potential for using the piggyBac transposon to engineer transgenic plants at specific genomic locations

“…In order to be competitive with nuclease mediated gene insertion in plants, 24,25 at least 2-5% of the embryogenic transgenic events should display the targeted insertion mediated by the piggyBac fusion transposase. The advantage to using piggyBac fusion transposase over a nuclease for gene insertion is that the transgenic plant would not need to be sequenced for unintended genetic changes that could occur with nucleases.…”

New developments in green biotechnology - an inventory for RIVM

“…Because the IPK1-gene is disrupted by a transgenic phosphinotricin acetyltransferase gene sequence insert (which mediates tolerance to the herbicide agent phosphinotricin (glufosinate) and is used for selection of HDR integration events), the maize plants with a reduced phytate trait still contain foreign sequences in the end product. HDR-mediated gene integration has also been used for sequential stacking genes into a so-called 'safe harbour' locus, which is a chromosomal location where genes can integrate and function in a predictable manner (Ainley et al 2013). An advantage of gene stacking at a single locus is that during subsequent crossings the stacked transgenes segregate as a single locus, which would simplify breeding of plant lines with stacked transgenes (Nandy et al 2015;Srivastava and Thomson 2016).…”