TRTech-PGR: NSF BSF - Recombining Allelic Diversity via Genome Editing for Dissecting Complex Plant Traits

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Global sustainability efforts rely on improvements in plant breeding technology particularly for crops of relevance for human consumption such as cereal crops. Wild relatives of domesticated plants harbor many of the genes that enable plants to become robust against stress. However, our ability to identify these genes and allocate their function to plant stress resilience has been difficult due to the low throughput of experiments needed to identify plant resilience genes. A main bottleneck limiting the throughput of testing crop genetic resilience is the ability to deliver biological molecules into plant cells, where the plant cell wall presents a largely impenetrable barrier for the introduction of the molecular biology tools needed for plant genetic mapping. In this project, nanoparticles will be developed to deliver genetic material into plant cells, with a focus on cereal crop barley. These nanoparticles will be chemically functionalized to deliver genome editing cargoes into plants, which will enable probing of specific plant genes in their relevance for plant stress tolerance. The project will seek to identify what genes enable cereal crops to be robust against environmental stress, and to leverage this knowledge to learn about stress tolerance in other crops of relevance for the global food supply. The research above will also support underrepresented undergraduate students as research trainees for the synthesis and characterization of nanomaterials and will develop a student workshop on nanomaterials science. The ability to study genotype to phenotype relationships in plants is critical for identification of plant biotic and abiotic stress genes. Specifically, crop wild relatives harbor genes that confer traits relevant for crop adaptation to climate change. Mapping genotype to phenotype relationships through functional genomics has benefitted greatly from the emergence of CRISPR genome editing technologies. However, the efficiency of genome editing in plants limits the utility of this method for functional genomics. In this project, nanoparticles will be developed for the physiochemical adsorption of CRISPR-based DNA plasmids and, separately, Cas9-gRNA ribonucleoprotein complexes. These nanoparticle-biomolecule cargo complexes will be tested for delivery and genome editing efficacy in model plants and in the cereal crop barley. The ability to deliver CRISPR plasmids in a non-integrating manner or to achieve DNA-free genome editing through Cas9-gRNA complex delivery would enable faster and more precise mapping of plant traits by avoiding transgene integration and the need for transgene segregation. Furthermore, the abiotic nature of nanoparticle-based DNA or protein delivery may enable facile genetic manipulation and functional genomics in other crops of relevance beyond those tested in the current work. In addition to the above efforts, this proposal will support training of undergraduate students from the Society for the Advancement of Chicanos and Native Americans in Science, and the development of a workshop to teach nanomaterials science to students from a broad variety of backgrounds.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.