Integration of PNA-mediated PCR and CRISPR/Cas13a for highly sensitive detection of EGFR T790M mutation in circulating tumor DNA.

Circulating tumor DNA (ctDNA) is characterized by low abundance and fragmentation, limiting the development of genetic variant detection technologies. In this study, we established a highly sensitive and specific assay by combining peptide nucleic acid (PNA)-mediated PCR clamping with CRISPR/Cas13a trans-cleavage detection. A PNA probe targeting the wild-type (WT) EGFR T790M allele was designed to suppress WT amplification during PCR, while minimally affecting mutant allele amplification. By combining the target specificity of Cas13a for mutant alleles with the WT-suppression capability of PNA-PCR, we achieved a dual-enrichment effect for mutant detection. When applied to EGFR T790M mutation detection, the assay reached an analytical sensitivity of 0.02%. We established a standard curve for T790M detection using cell-free DNA standards. Clinical validation in 20 plasma samples from lung adenocarcinoma patients demonstrated that the PNA-Cas13a assay achieved a diagnostic sensitivity of 93.3% (95% CI: 68.1%-99.8%) and a specificity of 100% (95% CI: 47.8%-100%), with detection concordance comparable to or improved over ARMS-PCR in this pilot cohort. The results suggest its preliminary diagnostic utility in liquid biopsy. In conclusion, the PNA-Cas13a assay enables sensitive and specific detection of EGFR T790M mutations in ctDNA, is readily adaptable to multiple gene loci, and holds promise for clinical monitoring of tumor drug resistance.