Development of an improved reporter system for assessing the activity of programmable RNA regulators



RNA-binding proteins (RBPs) play a key role in the control of gene expression through their regulation of mRNA half-life, localization, chemical composition, and translation. Aside from their roles in native biological regulation, RBP functions can also be leveraged to create synthetic RNA regulators by fusing functional components of RBPs to programmable RNA-binding platforms such as RNA-targeting CRISPR-Cas systems or CRISPR-Cas Inspired RNA-targeting System (CIRTS). However, a key bottleneck for developing programmable RNA regulators is a lack of robust reporter systems and methods to characterize their function. To develop better reporters to characterize RNA regulator activity we optimized a dual luciferase reporter system by selecting luciferase enzymes that maximize reproducibility and signal stability. Additionally, we demonstrated that untranslated regions (UTRs) from disease-relevant transcripts could be incorporated into the reporter mRNAs, both to test RBPs in more native sequence contexts, but also to evaluate RBPs ability to affect gene expression from an mRNA when directed to those sequences. We also demonstrate the flexibility of the reporter by tuning its translational efficiency, testing the reporter in multiple cell lines, and by testing validated RNA regulators that target several UTRs from disease-relevant gene targets. The new reporter system showcased here should provide a valuable method to characterize and develop novel RNA regulators for both synthetic biology and therapeutic purposes.


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