Nuclease-resistant DNA nanostructures for high precision plant genome engineering

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Background: CRISPR-Cas9 has brought the possibility precise genome engineering, yet barriers remain due to background random integration. This project will apply state of the art nanotechnology to improve the efficiency of CRISPR-mediated targeted genome modification of plant species. Directed improvement of crops is essential if we are to meet the demand for sustainable increased food and energy production against the challenges of climate change, limited land for cultivation and increased pressure on natural resources. Objectives: Nanotechnology allows the design of DNA structures that are resistant to nuclease digestion, through the formation of secondary and higher order complexes. These structures, lacking free duplex ends, will not be substrates for end-joining pathways that mediate random transgene integration, thereby promoting integration into homology directed pathways. This will enrich HR-mediated integration events in the generation of novel crop varieties. Novelty: This innovative and highly transformative approach will develop novel nanotechnology-based methodologies with potential high gain for genome engineering in plants, with application to other eukaryotic species.Timeliness: The project combines complimentary expertise of two leading groups working with CRISPR-Cas9 technologies and novel nanomolecular DNA structures with wide ranging applications across physical and life sciences. Bringing together these new technologies provides new possibilities for genome engineering and synthetic biology.Experimental approach: Plant cells will be transformed with a DNA end-free transgenes of looped DNA to provide a contiguous ring structure. More complex highly packed DNA structures will also be formed through the use of staple DNA moieties allows that confer high resistance to nuclease attack. The transgene will target the endogenous PROTOPORPHYRINOGEN OXIDASE gene to confer resistance to the herbicide butafenicil. Biolistic transformed plants will be further analysed to confirm gene targeting. A precise mechanistic understanding of integration pathway will be provided through a reverse genetic approach to identify the genetic determinants of site-directed insertion.