Abstract

The detection of single-base mutation in ctDNA with liquid biopsy is of great significance for the early diagnosis of pancreatic cancer. CRISPR-Cas system has attracted widespread attention in liquid biopsy due to its robust nuclease activity, extremely strong signal amplification capability and higher sensitivity. However, there is still a lack of a CRISPR-Cas-based method for single-base mutation detection that possesses high sensitivity and specificity while being simple to operate and highly practical. In this study, we innovatively designed a Double Blocker Integrated CRISPR-Cas14a (DBIC) system by introducing blocking RNA and blocking DNA. Our system can greatly improve the discrimination efficiency for base mismatches. Both theoretical analysis and experimental verification have confirmed that the limit of detection of this method can be as low as 0.0005%, which fully meets the requirements for early cancer screening. In the detection of actual clinical samples, we successfully achieved the identification of three highly prevalent hotspot mutation sites of pancreatic cancer using the DBIC system, and its effectiveness was verified by comparison with the results of NGS. Featuring simple operation, low cost and ultra-low detection limit, our system holds great promise for widespread clinical application.