(654f) Metabolite-Responsive Scaffold RNAs for Dynamic Cellular Control

Efficient biochemical synthesis requires the expression of both native metabolic pathways and heterologous enzymes that must be finely controlled in concert. CRISPR activation (CRISPRa) is a powerful tool for transcriptional control as it is programmable to act on a variety of gene targets and has been demonstrated to work in bacterial, yeast, and mammalian systems. However, a primary limitation of most CRISPR-based gene control systems is their static constitutive activation.

Here, we develop the metabolite-responsive scaffold RNA (MR-scRNA) to conditionally control CRISPR activation. In one flavor of CRISPRa, gene-specific recruitment of the transcriptional activator is enabled by translational fusion to a small viral coat protein (MCP) which binds to the 3’ end of a scaffold RNA (scRNA). We further modified the 3’ end of the scRNA with an RNA switch that conditionally forms an MCP-binding hairpin when the target metabolite is also bound. We took advantage of this cooperative binding event to create a MR-scRNA that conditionally recruits the transcriptional activator to the target gene. Using a theophylline-responsive MR-scRNA, we achieved >10-fold gene activation of a fluorescent reporter upon addition of the target metabolite. The MR-scRNA exhibited a dose-dependent response and is highly selective for the target metabolite over structurally similar molecules. The MR-scRNA also offers similar levels of gene activation with “PAM-less” dCas9 variants which greatly expands the number of possible targets that can be regulated. By employing a tryptophan-responsive MR-scRNA, we observed tryptophan-regulated CRISPRa and dynamic regulation of a five-gene biosynthesis pathway with our system indicating that MR-scRNAs can be used to dynamically regulate genes in response to physiologically relevant inputs. We envision that the simplicity of the MR-scRNA and its generalizable functionality will empower new opportunities for biosensing and dynamic regulation of cellular behavior.