CRISPR-Cas12a-integrated pregnancy test strip biosensors: Visual detection of telomerase and miRNA let-7a in cervical cancer diagnostics
Cervical cancer is a leading cause of female cancer-related mortality globally, and early screening based on reliable biomarkers is critical for improving prognosis. Telomerase (a key driver of cellular immortalization) and microRNA let-7a (a tumor suppressor with downregulated expression in cervical cancer) are well-validated diagnostic targets, but existing detection methods are hindered by complex procedures, high instrumentation costs, and reliance on specialized technical expertise-limiting their accessibility in resource-constrained settings. To address these limitations, we developed two novel CRISPR-Cas12a-integrated biosensors using commercially available pregnancy test strips (PTS) for instrument-free, visual readout. Both biosensors leverage a core signal mediator, probe 1 ("MB-ssDNA1-hCG"), which links CRISPR-Cas12a activation to visible color development on the PTS. The first Biosensor CRISPR-PTS-Telo detects telomerase activity in one-step without PCR: telomerase-generated (TTAGGG)n repeats activate Cas12a-crRNA1 complex, cleaving the probe 1 to release hCG, achieving a detection limit of 18 HeLa cells-comparable to sensitive laboratory assays. The second Biosensor CRISPR-PTS-let7a detects miRNA let-7a by first converting miRNA signals to Trigger DNA via Assister DNA and probe 2 ("MB-ssDNA2+Trigger"), activating Cas12a-crRNA2 complex, cleaving the probe 1 and inducing PTS coloration. This achieves a detection limit of 25.1 fM for let-7a. Validation with clinical samples (24 cervical tissues and 26 blood samples) confirmed their concordance with gold-standard methods (ELISA for telomerase, RT-qPCR for let-7a). These versatile tools hold significant potential as point-of-care testing (POCT) solutions to facilitate early, accessible cervical cancer screening.