Collaborative Research: EDGE FGT: Genome-wide knock-out mutant libraries for the microcrustacean Daphnia

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Mutating the DNA sequences of genes of interest is a valuable approach for understanding their biological functions in diverse organisms. In contrast to established model systems, large collections of gene knock-out mutants are not widely available in emerging model organisms. This limitation greatly diminishes the power of emerging model systems for making novel scientific discoveries. To address the issue, this project will develop a large collection of gene knock-out mutants using CRISPR gene editing in three species of the freshwater microcrustacean Daphnia (D. magna, D. pulex, and D. pulicaria), which is an emerging model system in ecological genomics, evolutionary genomics, and environmental genomics. The Daphnia knock-out mutants will provide unprecedented opportunities for the scientific community to characterize the biological functions of the genes. The project will also disseminate technical expertise for CRISPR gene editing in Daphnia through free workshops, targeting biologists at different career stages including undergraduates, graduates, postdoctoral researchers, and faculty. Furthermore, the project will develop an innovative genomics curriculum to enable hands-on research in the classroom and enhance the education experience of diverse undergraduate students. The plan is to knock out genes in Daphnia, including many candidate genes that may play critical roles in acclimation, adaptation, and evolution of these species. Individual knock-out mutant lines will be generated, maintained in the laboratory, and freely shared with the research community. Each mutant line will be whole-genome sequenced to characterize its genomic background. In collaboration with the global Daphnia research community, this project will evaluate the functional changes in knock-out mutants at both genomic and organism levels to validate the role of the genes in various environmental settings and to infer gene interaction and gene regulation networks. Lastly, a free, public-facing database will be developed to host the genomic and phenotypic data of the mutant lines, to promote and manage the distribution of Daphnia mutants, and to distribute experimental protocols and reagents.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.