Cationic LIposome capsulated CRISPR-Cas13a Kit (CLICK) for in situ detection of circulating PD-L1 mRNA in single EV towards monitoring of immunotherapy efficacy

Tumor immunotherapy has significantly improved cancer treatment, yet immune checkpoint inhibitors only benefit a subset of patients. A critical challenge is non-invasive detection of tumor PD-L1 mRNA, a key biomarker that dynamically reflects PD-L1 expression and correlates with therapeutic outcomes. Current methods are hindered by the instability of circulating mRNA and the need for invasive tissue biopsies. Herein, we introduce the Cationic LIposome capsulated CRISPR-Cas13a Kit (CLICK) for in-situ detection of circulating PD-L1 mRNA in extracellular vesicles (EVs). CLICK achieves direct target identification in 10 μL plasma within 2 hours without EV purification or RNA extraction, demonstrating a detection limit of 10 3 EVs/mL for PD-L1 mRNA. By integrating membrane fusion-mediated CRISPR delivery with nanoflow cytometry, CLICK resolves PD-L1 mRNA (+) EV subpopulations at single-vesicle resolution, revealing heterogeneous distributions. Clinically validated in 53 immunotherapy patients, CLICK accurately stratifies progressive disease (PD) from stable disease (SD) patients (AUC = 0.8236; 95% CI: 0.7054–0.9417; p= 0.0006) based on PD-L1 mRNA (+) EV percentages. This work establishes a rapid, blood-based paradigm for therapy response monitoring, advancing liquid biopsy through precise EV RNA characterization.