Collaborative Research: Synthetic CRISPR-Cas6 endonucleases for dynamic control of cellular phenotypes in yeast

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The overall goal of this project is to use the power of synthetic biology to build microbial factories for the production of high value chemicals. The collaborative research team will use an innovative enzyme colocalization/assembly strategy to boost production of selected chemicals in yeast. This project will also facilitate outreach activities to local high school teachers and students through existing programs available at the University of Delaware and University of California, Irvine.In nature, dynamic interactions between proteins play a crucial role in defining many cellular functions such as metabolism, cell signaling, transcription regulation, apoptosis, cellular targeting, and protein degradation. By controlling the spatial and temporal organization of these supramolecular complexes using a protein scaffold, cellular functions can be modulated in a highly dynamic manner for optimum efficiency. Understanding how these proteins interact holds the key to deciphering their roles in native cellular function and in creating new cellular functions for synthetic biology applications. In this project, the researchers propose a new transformative framework that combines the predictability of RNA hybridization and the ease of RNA processing, while offering reversible protein assembly on demand, to create dynamic enzyme cascades for proteasome-targeted protein degradation and for metabolic pathway regulation in yeast. The proposed research relies on a new and potent approach that enables specific processing and high-affinity binding to RNA transcripts using the naturally occurring CRISPR/Cas6 system. While the native function of Cas6 is to generate CRISPR RNAs (crRNAs) to guide the cleavage of DNA targets, the researchers will repurpose the Cas6 family proteins as a generalizable platform for site-specific RNA binding/processing and will demonstrate its utility to assemble dynamic enzyme cascades for synthetic biology and metabolic engineering applications in yeast. The expertise of the Chen and Da Silva labs will be combined to develop and implement the metabolons. State of the art synthetic biology and genetic tools will be used to accomplish four overall aims: (1) Dynamic protein assembly and disassembly by strand displacement, (2) Dynamic protein degradation using orthogonal Cas6 proteins, (3) Dynamic assembly of metabolons for substrate channeling, and (4) Combined metabolon assembly and protein degradation for increased polyketide biosynthesis.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.