(142b) Programmable Control of CRISPR-CAS9 Systems By Engineering Sgrna As Toehold-Switchable Riboregulators

Siu, K., University of Delaware
Chen, W., University of Delaware
Predictable control over gene expression is necessary for diverse applications in molecular biology, synthetic biology, and biotechnology. One of the most promising strategies to exert these types of control is the recently developed CRISPR interference (CRISPRi) and activation (CRISPRa) approach, which provides simple and highly effective RNA-based methods for targeted silencing and upregulation of transcription in bacterial and eukaryotic cells. While these current methods are capable of sequence-specific targeting, they lack the ability to integrate endogenous cellular signals as inputs for targeted regulation of gene expression. Here we present the design and construction of novel riboregulators by integrating toehold riboswitches into sgRNA scaffolds. These synthetic constructs are able to process specific information such as native metabolism and stress responses by taking advantage of highly predictable Watson-Crick base pairing mediated by RNA-RNA strand-displacement reactions. By exploiting the exceptional fidelity of these reactions, our toehold-sgRNA chimera can convert endogenous signals into cellular responses in the form of gene repression by CRISPRi. We demonstrate the programmability and adaptability of these engineered systems to control expression of multiple genes in E. coli with minimal cross-talks. These synthetic riboregulators represent a new class of RNA-based regulatory elements to facilitate targeted genetic perturbations and to guide cellular behaviors in a variety of context.