In Situ Amplified Mutational mRNA Imaging Using a Spatially Confined CRISPR Nanoplatform

Highly sensitive spatial analysis of RNA mutations is essential for understanding cellular heterogeneity and disease mechanisms. Herein, we developed an integrated CRISPR/Cas13a-based nanoprobe system for rapid detection of RNA in tissue sections (Integrated CRISPR/Cas13a-based RNA Rapid Detection, InCasRD). Unlike conventional "always-on" probes that rely on accumulated probe hybridization, InCasRD leverages the trans-cleavage activity of Cas13a to achieve spatially confined signal amplification and a high signal-to-background ratio (SBR). Using InCasRD, we achieved imaging of multiple target RNAs in tumor cells within 0.5 h of incubation, including mRNA (survivin), microRNA (miR-21), and circular RNA (circ1785). Furthermore, the engineered InCasRD system enabled mapping of RNA mutations, such as the EGFR L858R and ovarian tumor domain (OTUD) single-nucleotide variant (SNV, 23439980 G>T), in tumor tissue sections, thereby facilitating clear tumor boundary delineation. Collectively, InCasRD is a powerful, one-step tool for in situ RNA analysis with potential for diagnosis and precision medicine.