Stephen Novak

A modular gene targeting system for sequential transgene stacking in plants.

A modular, selection-based method was developed for site-specific integration of transgenes into a genomic locus to create multigene stacks. High-frequency gene targeting was obtained using zinc finger nuclease (ZFN)-mediated double-strand break (DSB) formation at a pre-defined target genomic location using a unique intron directly downstream of a promoter driving a selectable marker gene to facilitate homology between target and donor sequences. In this system, only insertion into the target locus leads to a functional selectable marker, and regeneration from random insertions of the promoterless donor construct are reduced on selection media. A new stack of transgenes can potentially be loaded with each successive cycle of gene targeting by exchanging the selectable marker gene using the intron homology. This system was tested in maize using the pat selectable marker gene, whereby up to 30% of the plants regenerated on Bialaphos-containing medium were observed to have the donor construct integrated into the target locus. Unlike previous gene targeting methods that utilize defective or partial genes for selecting targeted events, the present method exchanges fully functional genes with every cycle of targeting, thereby allowing the recycling of selectable marker genes, hypothetically for multiple generations of gene targeting.

A non-PCR-based Invader ® assay quantitatively detects single-copy genes in complex plant genomes

An accurate determination of gene copy number is critical to the success of a molecular breeding program involving both transgenic and non-transgenic plants. In this paper, we have described the application of a non-PCR-based technology, Invader®*, for determination of gene copy number and zygosity in plants. A biplex assay format detected both a target gene and an endogenous reference gene simultaneously from the genomic DNA. The ratio between the signals of the two genes in relation to known copy number standards of the same target gene allowed copy number determination. The linear range of the Invader assay was 1–4 copies per genome, but it can be accurate over a larger copy number range depending on the assay conditions. This technique was utilized for screening plants carrying low transgene copy numbers from a large number of events generated by plant transformation, and shown to produce results comparable to that of Southern blots. We have also utilized this technique to screen thousands of field-grown plants for zygosity determinations and obtained data that was over 98% accurate, thus proving that this assay can be used to improve the efficiency of a breeding program. Overall, the Invader assays proved to be reproducible, specific, applicable to any gene sequence and amenable to high-throughput screening.

RNAi targeting of rootworm Troponin I transcripts confers root protection in maize

Western corn rootworm, Diabrotica virgifera virgifera, is the major agronomically important pest of maize in the US Corn Belt. To augment the repertoire of the available dsRNA-based traits that control rootworm, we explored a potentially haplolethal gene target, wings up A (wupA), which encodes Troponin I. Troponin I, a component of the Troponin-Tropomyosin complex, is an inhibitory protein involved in muscle contraction. In situ hybridization showed that feeding on wupA-targeted dsRNAs caused systemic transcript knockdown in D. v. virgifera larvae. The knockdown of wupA transcript, and by extension Troponin I protein, led to deterioration of the striated banding pattern in larval body muscle and decreased muscle integrity. Additionally, the loss of function of the circular muscles surrounding the alimentary system led to significant accumulation of food material in the hind gut, which is consistent with a loss of peristaltic motion of the alimentary canal. In this study, we demonstrate that wupA dsRNA is lethal in D. v. virgifera larvae when fed via artificial diet, with growth inhibition of up to 50% within two days of application. Further, wupA hairpins can be stably expressed and detected in maize. Maize expressing wupA hairpins exhibit robust root protection in greenhouse bioassays, with several maize transgene integration events showing root protection equivalent to commercial insecticidal protein-expressing maize.

Trait stacking in modern agriculture: application of genome editing tools

Advances in plant transgenic technology in the 20th century overcame the major hurdle for transfer of genetic material between species. This not only enabled fundamental insights into plant biology, but also revolutionized commercial agriculture. Adoption of transgenic plants in industrial agriculture has reduced pesticide application, while bringing significant increase in crop yields and farmers9 profits. The progress made in transgenic technology over the last three decades paved the way mainly for simple single-gene insect and herbicide tolerance (HT) trait products. Modern agriculture demands stacking and pyramiding of complex traits that provide broad-spectrum insect and HT with other agronomic traits. In addition, more recent developments in genome editing provide unique opportunities to create precise on-demand genome modifications to enhance crop productivity. The major challenge for the plant biotech industry therefore remains to combine multiple forms of traits needed to create commercially viable stacked product. This review provides a historical perspective of conventional breeding stacks, current status of molecular stacks and future developments needed to enable genome-editing technology for trait stacking.