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Updated: Jul 12, 2026
Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids
Published on: June 8, 2019
Pan-Cancer Liquid Biopsy and Treatment Monitoring via a Split crRNA-Activated Label-Free CRISPR/Cas12a Platform for
Yixia Yang1, Qingming Pan1, Minling Liu1
1Guangdong Provincial Key Laboratory of Digestive Cancer Research, Department of Oncology, Department of Dermatovenereology, The Biobank, Scientific Research Center, The Seventh Affiliated Hospital, School of Medicine, Sun Yat-Sen University, Shenzhen 518107, China.
None:
Liquid biopsy based on circulating microRNAs (miRNAs) holds great promise for cancer diagnosis and treatment monitoring. However, the development of detection methods that are sensitive, specific, cost-effective, and compatible with diverse biofluids remains a challenge. Here, we report a sensitive and label-free detection platform, termed SCAN (Split crRNA-Activated CRISPR/Cas12a and Amplification Network), that integrates split CRISPR/Cas12a with catalytic hairpin assembly (CHA) for isothermal miRNA analysis. In this design, the target miRNA, serving as an alterable spacer RNA (sRNA), assembles with a conserved repeat RNA (rRNA) to reconstitute a functional full-length crRNA, activating the trans-cleavage activity of Cas12a. This cleaves a blocker probe and releases an initiator strand, which subsequently triggers a CHA cascade. The CHA reaction generates abundant G-quadruplex (G4) structures that bind specifically to N-methylmesoporphyrin IX (NMM), yielding a strong turn-on fluorescence signal. The optimized "signal-on" model achieved a detection limit of 2 fM for miR-21, offering approximately 5 orders of magnitude higher sensitivity than the basic split CRISPR/Cas12a system. The platform exhibited excellent specificity, capable of single-base mismatch discrimination, and could be readily adapted for detecting miR-128, miR-27a, and miR-155 through simple exchange of the double-stranded DNA activator. Importantly, by employing the label-free G4/NMM reporter, the cost of the signaling module was reduced by more than 45-fold compared to conventional dual-labeled probes. The SCAN platform reliably quantified miR-21 overexpression in colon cancer cell lines and robustly differentiated plasma samples from patients with multiple cancer types (colorectal, lung, cervical, breast, and thyroid cancers) from healthy individuals. Furthermore, it demonstrated utility in tracking treatment response through noninvasive urine analysis in prostate cancer and bladder cancer. This work establishes a sensitive, specific, low-cost, and versatile biosensing platform for miRNA-based liquid biopsy, holding strong potential for clinical diagnostic applications.
