Collaborative Research: Redirecting cellular metabolism via synthetic toehold-gated dCas9 regulators

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Cellular metabolism is capable of highly specific and efficient chemical synthesis at mild temperatures and pressures far beyond the capability of most synthetic chemical routes. Engineering specific pathways can be used to further improve the range of compounds that can be synthesized but it is a major challenge to achieve commercially viable productivity. To maximize productivity, it is crucial to fine-tune pathway fluxes. The goal of this project is to develop a new transformative approach to modulate cell metabolism based on endogenous cellular information. An emerging strategy is the use of regulators that provide dynamic control of pathway fluxes. A recently discovered modified CRISPR based tool offers a unique approach for DNA targeting and transcriptional regulation. These new generation of regulators can be used for dynamic gene repression and activation for many synthetic-biology and metabolic engineering applications. In addition to the scientific advancements, this project will help train graduate students through the integration of principles from protein engineering, synthetic biology, and cellular physiology. Outreach activities to local high school teachers and students through existing programs available at the University of Delaware and Rensselaer Polytechnic Institute are also planned.The goal of this project is to develop a new transformative approach to modulate cell metabolism based on endogenous cellular information. In particular, a new generation of toehold-gated dCas9 regulators governed by conditional sgRNA structures that are activated by toehold-mediated strand displacement will be created to provide simultaneous, orthogonal, and autonomous control of cellular metabolism. Because dCas9-based regulators are governed by a structurally defined single guide RNA (sgRNA) structure, it is easy to envision that conditional sgRNA structures can be created that are activated by endogenous mRNAs based on toehold-mediated strand displacement. This new framework to design toehold-gated dCas9 regulators responsive to any endogenous mRNA will lay the foundation as a new transformative approach for implementing dynamic control of cellular metabolism. The ability to modulate metabolism based on endogenous cellular information in optimizing the production of numerous products in yeast will be established. The long-term goal is to combine the knowledge gained from this project toward the design of dynamic and autonomous cellular control for any metabolic pathway of interest.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.