Accurate and rapid detection of RNA biomarkers is crucial for early disease diagnosis and infectious disease management. Although RT-qPCR remains the gold standard, its utility for point-of-care testing is limited by run-to-run variability and dependence on specialized instrumentation. Conversely, colorimetric assays offer operational simplicity but generally fall short of the sensitivity and specificity demanded for reliable applications. To bridge this gap, we present an innovative cut-and-bind colorimetric RNA detection platform that synergistically integrates the sequence-specific programmable cleavage fidelity of CRISPR/Cas12a with zinc finger protein-mediated gold nanoparticle assembly (termed CasFinger-Gold). Strategically engineered ssDNA-dsDNA hybrid overhang probes sharpened target discrimination while minimizing off-target background, ensuring robust readouts within complex biological matrices. When coupled with reverse transcription-recombinase polymerase amplification, the CasFinger-Gold system achieved visually interpretable RNA detection with a limit of detection of 1.8 × 103 copies μL-1 for HCoV-OC43 viral RNA and 0.4 ng total RNA μL-1 for the long non-coding RNA PCA3. The platform further enabled direct, rapid (<120 min), purification-free PCA3 detection from cell-derived extracellular vesicles (EVs) at clinically relevant vesicle inputs (down to ∼106 EVs μL-1). Notably, no cross-reactivity was observed against four phylogenetically distinct respiratory viruses or in PCA3-negative cell lines. By delivering instrument-free visual readouts comparable to RT-qPCR, CasFinger-Gold represents a versatile diagnostic modality poised to expand the molecular diagnostic toolbox toward accessible point-of-care solutions.
