Universal and highly accurate detection of circulating tumor DNA mutation in non-small cell lung cancer based on CRISPR/Cas12a system

Circulating tumor DNA (ctDNA) is a promising biomarker for real-time, minimally invasive diagnostics and monitoring in patients with non-small cell lung cancer (NSCLC), especially when representative tissue biopsies are not available. However, the current methods for ctDNA detection are cumbersome and expensive. While advanced CRISPR/Cas-based assays offer advantages of simplicity, low cost and high sensitivity, their application for ctDNA detection is restricted by the requirement of a protospacer adjacent motif (PAM) near the mutation site and off-target cleavages (i.e., false-positive results) due to the extreme similarities between the mutant and wildtype sequences, especially single nucleotide variants. Herein, we propose a novel strategy comprising recombinase polymerase amplification (RPA) and CRISPR/Cas12a to detect ctDNA with high universality and accuracy. The use of artificially inserted PAMs by modified RPA primers or suboptimal PAMs unlocks the PAM restriction; introducing single- or double-base mismatches in CRISPR RNA effectively reduces the off-target effects and improves the specificity to single-base resolution. Under optimized conditions, this method detected ctDNA mutations with a limit of detection at 100 aM and identified mutations down to 0.02% variant allele frequency in 50 min, requiring only isothermal control. We successfully applied this method to multiple clinical samples of NSCLC and the results were validated using real-time polymerase chain reaction analysis. In summary, we established a rapid, sensitive, universal and highly accurate method for ctDNA detection that has great potential application in the early diagnosis, therapy guidance and prognosis prediction of NSCLC.