Versatile and dynamic gene regulation using synthetic non-coding RNAs

Bacteria hold great potential as a platform to produce hundreds or thousands of natural products derived from organisms across the tree of life, which can be applied towards a number of diverse applications, including environmental remediation, commodity chemical synthesis, and health and medicine. Accessing these products is solely dependent on our ability to reprogram bacteria through the design and implementation of synthetic devices. Recently, RNA regulators have emerged as a reliable tool for the construction of devices that can create desired patterns of gene expression. These RNA regulators are highly structured molecules that exhibit diverse functionalities, including sensing, regulatory, and scaffolding activities. In this work, we combine the CRISPR system and synthetic antisense RNAs in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Next, we demonstrate an integrated genetic circuit compiled from the STAR system and synthetic antisense RNAs to activate or deactivate a target gene. Furthermore, two A AND NOT B logic gates are constructed and tested in the same cell using the combined STAR and antisense RNA system to demonstrate sophisticated multi-gene regulation. Finally, a data-driven model is developed for predictable and tunable antisense RNA-mediated repression. This predictive model is validated in a number of different genetic contexts and organisms. Together, this work establishes a methodology for integrating multiple RNA regulators to modulate multiple genes’ expression and provide a generalizable model that enables predictable antisense RNA-mediated gene repression in diverse bacterial species