GM plants 2.0: Can DNA nanotechnology enhance plant genome engineering?

Abstract

Gene targeting (GT) is a method of precise genome engineering that can facilitate crop improvements that are central to meeting growing global food demands. Yet GT frequencies are very low in higher plants, mainly due to the low frequency of homologous recombination (HR) upon which GT relies, hindering realisation of the technology’s potential. Despite advancements, routine GT frequencies remain ~ <1% and random integration of donor DNA and GT machinery causes problematic off-target effects; therefore, further improvements in GT frequency and accuracy are required. Here, a novel approach to address these problems is described, based on the application of DNA nanotechnology to GT in plants. In this approach random integration of the GT template will be prevented by concealing the free dsDNA ends of the repair template with nanostructure ‘caps’, as we propose that it is the free dsDNA ends that serve as the substrate for random integration. Here I report that capped structures containing a GFP reporter gene have been successfully transformed into plant cells via Agrobacterium and GFP expression detected on confocal microscopy. This demonstrates that these structures get into the plant cell nucleus and do not hinder enzymatic access to the gene, an important milestone in developing this technology. To test the nanostructure’s ability to improve GT efficiencies and reduce off target effects, an assay was established to measure GT rates with or without nanostructures. GT events have been successfully visualised by GFP expression in plant cells using confocal microscopy and flow cytometry. Next steps are to assay the rates of random integration using the nanostructure caps via qPCR-based assays and to design, build and test the next generation of DNA nanostructures for prevention of random integration using DNA origami nanotechnology as a transgene delivery vehicle. Overall, this project brings together two exciting technologies to address the need for continued improvement in GT frequencies and preventing random integration. This will facilitate the improvement of plant genome engineering tools that can deliver vital improvements to crops required to bring sustainable agriculture into the 21st Century.