(619a) Development of a Novel RNA-Sensing Spatiotemporal Gene Regulation Program for Eukaryotic Systems

To effectively probe gene function and reprogram cell regulatory networks, it is critical to have technology platforms that provide precise and accurate targeting of genes. The use of CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) for targeted silencing or upregulation of transcription, respectively, is currently one of the most utilized technologies for predictable control over gene expression. The CRISPR system allows for sequence-specific targeting of genes but inherently lacks the ability to incorporate useful endogenous signals for spatiotemporal control of gene expression. Here we present the design and further characterization of a class of riboregulators through the incorporation of toehold riboswitches into sgRNA scaffolds. This artificial circuit is able to detect the presence of specific RNA and switch on transcriptional level gene regulation through RNA-RNA strand displacement reactions, which are governed by predictable Watson-Crick base pairing. These synthetic constructs can be programmed to process specific information within the cell including changes in native metabolism and stress responses. We demonstrate the programmability and adaptability of these engineered systems to control gene expression in eukaryotic systems with minimal infidelity. These synthetic riboregulators can be applied in a variety of contexts within synthetic biology and the potential to function as an RNA-based master regulator for autonomous cellular control to direct specific phenotype in CHO cell cultures.