Single-Molecule Imaging of Endogenous RNA Using a Miniaturized and Circularly Permuted CRISPR-Cas13 Degron-Masking System CDegSR.

Visualizing the spatiotemporal dynamics of endogenous, unmodified RNA within living cells is crucial for understanding gene regulation. Yet, existing techniques are often hindered by high background fluorescence and the steric hindrance posed by CRISPR effectors. To address this, we developed CDegSR (CRISPR-mediated Degron-based Signal Restoration), a compact, fluorogenic system for high-contrast RNA imaging at single-molecule resolution. By miniaturizing dPspCas13b to its essential recognition lobe and employing circular permutation, we repositioned the protein termini to enable target-dependent masking of a potent degron. This approach promotes rapid proteasomal degradation of effectors not bound to targets while selectively stabilizing those attached to target RNA. Using CDegSR, we achieved single-molecule tracking of endogenous POLR2A transcripts, revealing that nuclear crowding and the translation machinery are the main physical constraints on mRNA mobility. CDegSR provides a versatile, low-perturbation toolkit for dissecting the transcriptomic lifecycle at single-molecule resolution.