Intelligent guide RNA: dual toehold switches for modulating luciferase in the presence of trigger RNA

Abstract

Abstract The CRISPR system finds extensive application in molecular biology, but its continuous activity can yield adverse effects. Leveraging programmable CRISPR/Cas9 function via nano-device mediation effectively mitigates these drawbacks. The integration of RNA-sensing platforms into CRISPR thus empowers it as a potent tool for processing internal cell data and modulating gene activity. Here, an intelligent guide RNA—a cis-repressed gRNA synthetic circuit enabling efficient recognition of specific trigger RNAs—is developed. This platform carries two toehold switches and includes an inhibited CrRNA sequence. In this system, the presence of cognate trigger RNA promotes precise binding to the first toehold site, initiating a cascade that releases CrRNA to target a reporter gene (luciferase) in this study. Decoupling the CrRNA segment from the trigger RNA enhances the potential of this genetic logic circuit to respond to specific cellular circumstances, offering promise as a synthetic biology platform.