Engineered crRNA Drives RPA‐T7‐CRISPR/Cas14a Cascade for Ultrasensitive Detection of ctDNA PIK3CA H1047R
Yuanyuan Yu, Mengru Jin, Weiguang Yuan, Yajie Gong, Siwei Li, Xuquan Qin, Jianxun Hou, Jialin Liu, Siyu Liu, Hui Li, Yijun Chu, Yingjie Wang, Youxue Zhang, Fang Fang, Wenhui Hao, Yuling Gu, Qinchen Fan, Jing Lin, Da Pang, Xianyu Zhang
Abstract
The early detection of circulating tumor DNA (ctDNA) at mutant allele frequencies below 0.1% remains a critical challenge, significantly impeding therapeutic decision-making. To address this limitation, TIDE-Cas14a-an innovative CRISPR/Cas14a-based duplex detection system is developed that integrates recombinase polymerase amplification (RPA) with T7 exonuclease-mediated strand displacement. By strategically engineering crRNAs with synthetic mismatches, the platform achieves single-nucleotide resolution, enabling specific discrimination of the PIK3CA H1047R (c.3140A>G) variant from other mutant subtypes and wild-type sequences at a detection limit of 0.01% with attomolar sensitivity. The system leverages T7 exonuclease's 5'→3' digestion to convert RPA amplicons into single-stranded targets, thereby activating Cas14a without requiring thermal cycling. Furthermore, clinical validation using 32 breast cancer patient samples demonstrated that TIDE-Cas14a achieves 100% sensitivity and specificity, comparable to droplet digital PCR. When deployed on a low-cost digital microfluidic chip, the assay completes ctDNA profiling within 60 min at 37 °C, effectively bridging the gap between complex laboratory testing and point-of-care diagnostics. The work repurposes the CRISPR/Cas system's inherent specificity constraints as a precision oncology tool, establishing a scalable platform for early cancer detection and therapeutic monitoring.