PlantSynBio: Development and application of a versatile CRISPR platform for simultaneous gene editing and transcriptional regulation in plants

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Plant synthetic biology is an emerging field that combines engineering principles with plant biology. To speed up the design-test-build-learn cycle in synthetic biology, new tools need to be developed in plants. In recent years, gene editing tools for altering gene sequences and gene regulation tools for altering gene expression have both been developed in plants. However, tools that allow for achieving gene editing and gene regulation at the same time are not yet available to the plant research community. To fill in this major technology gap, a toolbox capable of inducing simultaneous gene editing, gene activation and gene repression will be developed. These tools will be demonstrated in rice, a major crop that feeds a third of the world population. Demonstrations will include the development of high-yield Golden Rice, which provides a promising solution for treating vitamin A deficiency in humans, and the development of herbicide-resistant proanthocyanidin-biofortified rice with high nutrition value. This project will ignite and amplify community interest in applying new synthetic biology tools for next-generation metabolic engineering to make better crops that are high-yield, more nutritious, disease-resistant and more resilient in a changing climate. Training future scientists, especially from under-represented minority groups, is a core mission of this research project. The development of CRISPR-Cas based genome engineering tools has greatly accelerated crop breeding and metabolic engineering. To unleash the potential of plant synthetic biology and systems biology, innovative tools are required for simultaneous manipulation of the plant genome and transcriptome. This proposed project aims to develop and apply an easy-to-use, highly efficient and multifunctional CRISPR-Cas9 platform for simultaneous and orthogonal gene editing, transcriptional activation and repression in plants. The platform will be demonstrated by generating biofortified rice with concurrent engineering of other desirable traits. The project aims are to (1) develop novel CRISPR systems for simultaneous gene editing and transcriptional regulation in plants, (2) apply a simultaneous gene editing and activation system to engineer high-yield Golden Rice, and (3) apply a simultaneous gene editing, activation and repression system to engineer herbicide-resistant proanthocyanidin-biofortified rice. Although rice is used for technology development, the tools are compatible with applications in other plants. The vectors developed for this multifunctional CRISPR-Cas9 platform will be made available to other researchers so that they can be widely adopted by the plant research community for basic and translational research. The broader impacts of this project include training for undergraduate students at University of Maryland at College Park, University of California at Davis and Howard University at Washington. The project also provides STEM training to students from Montgomery Blair High School and Eleanor Roosevelt High School.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.