CRISPR-Cas12a based detection of EGFR gene mutation in cell free DNA for early diagnosis of Non-Small Cell Lung Cancer (NSCLC)

Abstract

Detecting EGFR mutations in circulating cell-free DNA (cfDNA) is essential for personalizing treatment and real-time monitoring of non-small cell lung cancer (NSCLC), especially when tissue biopsies are not an option. Conventional methods for identifying these mutations in cfDNA are limited and often require extensive laboratory set-up. The CRISPR-Cas system, notably the Cas12a variant used in the DETECTR method, presents a groundbreaking approach for highly sensitive and specific mutation detection in biological fluids, significantly advancing diagnostic capabilities. In this study, we present a novel, rapid, and precise method combining Recombinase Polymerase Amplification (RPA) for DNA amplification with a CRISPR-Cas12a fluorescence assay to detect EGFR mutations. This technique achieves attomole-level sensitivity without cross-reactivity, functions near 37°C, and delivers results within an hour, making it highly suitable for clinical applications in precision oncology. To further enhance its utility for point-of-care diagnostics, we integrated Cas12a with lateral flow-based detection, achieving single-copy detection sensitivity. In a clinical trial involving 57 NSCLC patient samples, this method successfully identified EGFR mutations, with results consistent with those obtained via PCR. The CRISPR-Cas12a based lateral flow assay demonstrated exceptional sensitivity and specificity, even detecting dual mutations simultaneously on a single test strip. These results position the CRISPR-Cas12a detection system as a straightforward, highly sensitive alternative to PCR, with significant potential for point-of-care applications in detecting cfDNA mutations.