CAREER: Nanoparticle-mediated genome engineering of plants and plastids

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Genetic engineering of plants is at the core of sustainability efforts, drug synthesis, and agricultural engineering. The predominant bottleneck facing efficient plant genetic engineering is biomolecule delivery into plant cells through the rigid and multi-layered cell wall. This constraint limits the species of plants that can be genetically engineered. In this project, nanoparticles will be developed to deliver genetic material into the plant chloroplast, which is a gene-containing organelle responsible for photosynthesis. The project will first focus on the development of molecular biology tools and nanoparticles that can traverse the plant cell wall and deliver biomolecules into plant chloroplasts. Next, the project will test efficiencies of nanoparticle-based chloroplast delivery tools and compare them with conventional tools, with the goal of achieving higher genetic transformation efficiencies. The project will conclude by incorporating genome-editing biomolecules into nanoparticles to demonstrate genome editing in the chloroplast. The potential impact of the proposed approach will be to enable genetic engineering of the plant chloroplast, thus enabling the discovery and study of chloroplast genes and the potential use of chloroplasts for producing valuable protein products. For outreach and education, an international virtual conference will be developed that brings together scientists, policymakers, and the public to discuss scientific topics and the dissemination of literature in the media. An undergraduate course will be developed to expose undergraduate students to the peer-review process and hypothesis-driven research, and high school teachers will be brought into the laboratory for hands-on research training during the summer. These efforts will increase scientific literacy in the public, universities, and schools.Despite the importance of creating transgenic plants, plant systems, particularly non-model plant species, remain difficult to genetically transform. The dominant bottleneck to plant transformation is the delivery of the molecular biology tools needed for plant transformation or gene silencing: DNA, RNA, and protein, across the plant cell wall, particularly for chloroplast-targeted genetic manipulation studies. In this project, nanoparticles will be developed and functionalized to adsorb and deliver plasmid DNA to plant chloroplasts in a species-independent and high-efficiency manner. To this end, in Objective 1, genome editing plasmid DNA vectors will be developed and delivered to model plant leaves and tissues to test genome editing efficiencies and to confirm a lack of transgene integration. This objective will confirm the ability of nanomaterials for chloroplast genetic transformation with efficiencies that surpass those of conventional tools. Objective 2 will subsequently develop, and codon optimize a luciferase-based plasmid reporter system to enable expression of luciferase in plant chloroplasts, with a goal of exceeding transformation efficiencies achievable with biolistic DNA delivery. Lastly, in Objective 3, a genome editing plasmid will be developed based on knowledge gained in Objectives 1 and 2 to target endogenous chloroplast genes in both model and non-model plant species. The ability to genetically manipulate chloroplasts, particularly in non-model plant species, will broaden the range of plants that can currently be studied. Genetic manipulation of the plant chloroplast genome will in turn uniquely enable the study of genes coding for most photosynthetic proteins, especially in non-model plants. This project will also leverage features unique to the chloroplast including a lack of gene silencing pathways, to enable high efficiency expression of transgenes. This latter feature makes chloroplasts an attractive target for biosynthetic production of commodity chemicals, drugs, and even larger protein products such as antibodies and biologics. Orthogonally, because the proposed approach is non-pathogenic in nature, the broader impact of this approach could enable more seamless regulatory oversight of transformed plant tissues in crop species. This project will also develop three efforts to increase scientific literacy. In the first effort, an international virtual conference will be developed that brings together scientists, policymakers, and the public to discuss scientific topics and the dissemination of literature in the media. In the second effort, an undergraduate course will be developed to expose undergraduate students to the peer-review process and hypothesis-driven research. In the third effort, high school teachers will be brought into the laboratory for hands-on research training during the summer. These efforts will increase scientific literacy in the public, universities, and schools.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.