Prostate cancer (PCa) diagnostics urgently require non-invasive methods with improved specificity beyond PSA testing. While PCA3 RNA and oncogenic microRNAs (e.g., miR-21) are promising biomarkers, their clinical utility is hindered by the need for complex RNA amplification in current assays. We developed a CRISPR/Cas12a-mediated liquid biopsy platform for simultaneous, amplification-free detection of PCA3 and miR-21 in urine and blood, aiming to enhance diagnostic accuracy through multi-analyte profiling.

A multiplexed split CRISPR/Cas12a system was engineered with dual crRNAs targeting PCA3 RNA and miR-21, coupled with spectrally distinct fluorescent reporters. The platform was validated using 51 blood (plasma) and 58 urine samples from 38 PCa patients and 20 controls. Performance metrics included sensitivity, specificity, AUC, and biomarker concordance with tissue biopsy (κ score).

Dual biomarker analysis significantly improved diagnostic accuracy versus single-target detection. In urine, combined PCA3 + miR-21 testing achieved 94.7% sensitivity (95% CI: 89.2–97.7%) and 95.0% specificity (95% CI: 93.4–99.6%) (AUC=0.95), outperforming PCA3-alone (89.5% sensitivity, 85.0% specificity, AUC=0.88) and blood-based testing (92.1% sensitivity, 90.1% specificity, AUC=0.91). The platform detected both targets at 10 copies/μL within 2.5 hours, with strong agreement to biopsy (κ=0.89).

This CRISPR/Cas12a platform enables rapid, multiplexed detection of urinary PCA3 and miR-21 without amplification, demonstrating synergistic diagnostic power for PCa. The dual-RNA approach addresses tumor heterogeneity challenges, offering a robust tool for non-invasive screening and molecular stratification.