EDGE: High Efficiency Identification of Products of Homologous Recombination in Plants as a Tool to Test Gene Function

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This project will develop a new method to facilitate and accelerate the identification of successful products of homologous recombination (HR) in crop plants. This method is based on advances in 'genome editing', a revolutionary technology that promises the ability to introduce genetic improvements or novel functionality to genomes where genetic resources are currently limited. Applications in plants that require replacing an endogenous piece of DNA with another DNA sequence are possible but are very inefficient due to very low rates of HR. The technology developed in this project is designed to overcome this bottleneck, with the aim to advance organismal biology by greatly facilitating the testing of gene function through direct modification of a broad range of genes of interest. Such advances will contribute to improvements in the understanding of the genomes-to-phenomes relationship. This could, for example, accelerate the development of plants with improved resistance to pathogens, or enhanced crop yields. The project will carry out experiments to further develop, test, validate and improve this technology and its application, first testing the components of the system, then applying what is learned to single and multiple targets. The work will test the system in cassava and foxtail millet, as well as in model flowering plants. Broader impacts of the project include the wide dissemination of the method for the improvement of crop plants and other species, plus the training of students via integration with long-running and successful undergraduate internship program. The project will focus specifically on techniques to facilitate and accelerate the identification of successful products of HR by reporter-based methods using transcriptional activation. The approach combines insertion of a repair template (containing the desired genome edit and a selectable marker) with methods to rapidly find a desirable, rare HR event - the proverbial "needle in a haystack". Data from cassava demonstrates that this is an effective method for developing molecular and genomic tools for orphan crops and those lacking robust genomic resources. The work will be done in cassava, Arabidopsis, and Setaria as the primary species for analysis, because a demonstration in these species would support broad utility in eudicot and monocot species, including most crops. The questions to be addressed by this project include: 1) whether the efficiency of HR can be improved by focusing on selection of the products; 2) whether HR-based approaches be used to successfully tag non-coding RNAs such as microRNA and tasiRNA precursors; and, 3) whether it is possible to develop efficient processes for high-throughput, library-based knock-ins. The project will develop an approach for a key functional genomic tool (improved selection of products of HR) to enable direct tests of hypotheses about gene function in diverse organisms. Project members will be trained broadly in plant biology and genomics, gene function and functional genomics, and computational methods. All resources generated in this project will be made available for use. Specifically, constructs will be deposited in nonprofit global plasmid repositories and web-based tools will be designed to enable users unfamiliar with genome editing to successfully design custom components of SureFire for application to their organism(s) of study.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.