Integrated one-pot RPA-CRISPR/Cas13a platform enables ultrasensitive and field-deployable JAK2 V617F detection for myeloproliferative neoplasm diagnosis.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic malignancies driven by mutations like JAK2 V617F, a constitutively activating variant diagnostic for polycythemia vera and subsets of essential thrombocythemia and primary myelofibrosis. Sanger sequencing is the gold standard for detecting JAK2 mutations, but it faces limitations in terms of cost, sensitivity, and portability. CRISPR diagnostics generally requires preamplification, which increases the risk of contamination. There remains an unmet need for field-deployable, rapid MPNs diagnostics in resource-limited settings. We developed ONE-CASPR, a one-pot RPA-CRISPR/Cas13a system enabling ultrasensitive (0.1 % mutant allele frequency), rapid (30 min), portable JAK2 V617F detection. The platform synergizes recombinase polymerase amplification (RPA) with Cas13a trans-cleavage activity in a single-tube reaction at 37 °C, executed via a miniaturized wireless analysis device (CPod) to eliminate cross-contamination while preserving accuracy. Clinical validation across 36 patient samples demonstrated 100 % concordance with real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) in both sensitivity and specificity. ONE-CASPR provides a rapid, sensitive, field-deployable, and user-friendly solution for MPNs point-of-care diagnosis in resource-limited settings, with broad molecular screening application potential.