(146a) Construction of Programmable Genetic Circuits through Generalizable RNA Regulators and Crispr Interference

The past decades have witnessed the power of RNA regulators for precise and orthogonal gene expression control. Although gene regulation studies have focused more on protein regulators than RNA regulators, RNA regulation has many advantages over protein regulation. RNA regulators are easier to design than protein regulators, due to the simplicity of base-pairing rules and our increased understanding of RNA folding and functions. Additionally, RNA regulation is faster and has a lower metabolic burden than protein regulation because regulatory RNAs are shorter than regulatory proteins, and RNA regulators do not undergo the cellular resource-intensive process of translation. Still, synthetic RNA regulators for precise and tunable gene expression control are in great demand, and we aim to provide generalizable RNA regulators that allow for rational reprogramming of cellular functions. To this end, we have employed the CRISPR interference system (CRISPRi) from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) to repress or derepress target genes in a programmable manner. Specifically, we demonstrated that the repressed target output by the CRISPRi system can be derepressed by activating transcription of asRNAs that sequester their target small guide RNAs (sgRNAs). Our regulation system is unique in that it combines dCas9-sgRNAs with asRNAs, which can control the dynamics of gene expression. Furthermore, these RNA-based genetic circuits have been optimized by varying design parameters (e.g., the hybridization free energy of the sgRNA-asRNA complex), and tunable levels of derepression have been achieved (up to 95%). These RNA regulators can also target multiple genes orthogonally, allowing for multiplex gene regulation. To our knowledge, this is the first demonstration of a tunable RNA circuit that integrates multiple types of RNA regulators in bacteria. This new system will allow for rational reprogramming of cellular processes, facilitating diverse synthetic biology applications. We will present progress towards development of the design principles for generalizable RNA regulators.