One-pot SP-EXPAR-integrated CRISPR/Cas14a assay for highly specific and versatile detection of ctDNA mutations in lung cancer patients.

The highly specific and versatile detection of KRAS mutations in circulating tumor DNA (ctDNA) from plasma has critical clinical implications for non-small-cell-lung cancer (NSCLC). However, conventional isothermal amplification methods suffer from poor single-base discrimination, while CRISPR-12a-based detection is highly protospacer adjacent motif (PAM)-dependent. To address these challenges, a one-pot self-primer isothermal exponential amplification reaction (SP-EXPAR) combined with a CRISPR/Cas14a assay was developed for detecting KRAS G12C and G12D. Two synergistic strategies were devised to ensure high specificity: first, a carefully designed hairpin probe that permits selective amplification of mutant over wild-type sequences through differential binding affinity; second, optimization of the Cas14a sgRNA seed region, with the mutation positioned at the 11th nucleotide for stringent target recognition. The assay is further distinguished by a physical separation design, in which the Cas14a reagents are pre-loaded into the tube cap and mixed with the amplification products only after SP-EXPAR completion. This assay enables KRAS G12C detection within 1 h, with a limit of detection of 81.9 aM (0.1% mutation percentage) and a dynamic range from 100 aM to 1 nM. Furthermore, this assay further demonstrates its programmability and was successfully applied to detect KRAS G12D with comparable performance. In detecting 42 clinical samples, this assay demonstrated 100% sensitivity and 100% specificity compared with DNA sequencing. This approach holds great potential in disease diagnosis.