Extracellular vesicle (EV)-mediated communication is tightly regulated by local pH, which governs vesicle biogenesis, cargo release, and membrane fusion. Accurate and context-specific pH sensing is therefore crucial for elucidating EV function and disease-associated microenvironmental regulation. Here, we present a tetrahedral DNA framework (TDF)-orchestrated dual-CRISPR system that integrates orthogonal Cas12a and Cas13a nucleases for target-activated, ratiometric pH detection at lipid membranes. By exploiting the distinct pH-activity profiles of Cas12a (optimal pH ∼ 8.5) and Cas13a (optimal pH ∼ 7.2), combined with their complete substrate orthogonality, we constructed a self-calibrating nanosensor featuring equimolar coassembly of both nucleases and their corresponding fluorogenic reporters at the four vertices of a TDF. The well-defined tetrahedral geometry ensured reproducible molecular organization and stable fluorescence output, eliminating variability inherent to conventional single-fluorophore probes. The sensor exhibited quantitative assembly fidelity and robust pH responsiveness across physiological ranges. Importantly, the Cas module can be programmed for conditional activation, enabling pH sensing only upon recognition of disease-associated biomarkers. Using miR-146a, a regulatory microRNA enriched in EVs implicated in inflammation and cancer progression, as a model target, we demonstrated target-gated pH monitoring on cell-derived exosomes and during liposome fusion events. This work establishes a versatile and generalizable platform for programmable, ratiometric sensing at biomembrane interfaces, offering new opportunities to probe EV-mediated intercellular communication and dynamic microenvironmental regulation.
Fang et al. (2026) studied this question.