Switched Fluorogenic CRISPR-tDeg System for RNA Imaging in Living Cells

RNA displays significant heterogeneity in its structures, dynamics, and functional roles within cells. CRISPR-based imaging methods provide a powerful approach to visualize RNA molecules in living cells. However, the expression of constitutive fluorescence modules in traditional CRISPR-based tools always produce high background and nonspecific signals. To address these challenges, we developed a switched fluorescent CRISPR-tDeg (CtDeg) system that minimizes background for RNA imaging in living cells. This CtDeg system consists of a signal module-deactivated Cas13 protein (dCas13)-degron complex and an engineered switched sgRNA. The signal module-fused complex is degraded unless it binds to the engineered sgRNA, which is activated upon targeting the desired RNA. This interaction will generate a stable signal module-dCas13: sgRNA complex, enabling specific imaging of RNA in living cells. Using the CtDeg system, we visualized the movement and assembly dynamics of paraspeckles at the RNA level. Additionally, we tracked SARS-CoV-2 genome dynamics at the early stage after viral invasion for the first time, providing direct imaging data that demonstrate SARS-CoV-2 infection up-regulates the expression levels of the disease-associated NEAT1 gene, thereby filling a gap in this research area. The CtDeg system represents a powerful tool for specific imaging of RNA to reveal the underlying functions and interactions within living cells.