ScholarWorks@성균관대학교

초록

Simple Summary Many efforts have been made to detect pathogenic mutations in cell-free circulating DNA (cfDNA) derived from blood, which offers a less invasive and more practical alternative to tissue biopsy. However, the low abundance of mutated DNA in blood samples makes detection by high-throughput sequencing challenging. In this study, we developed a highly sensitive CRISPR/Cas12a-based diagnostic system that selectively removes wild-type (WT) DNA to enrich rare mutant DNA, enabling reliable mutation detection through PCR. Applying this approach to cfDNA from 48 non-small-cell lung cancer (NSCLC) patients, we targeted two clinically meaningful EGFR mutations: L858R and exon 19 deletion. Our system successfully detected all 7 L858R-positive cases and 6 of 11 exon 19 deletion-positive cases, consistent with prior tissue biopsy results. These findings demonstrate that our CRISPR/Cas12a-based system enables sensitive and selective detection of low-frequency mutations in cfDNA, supporting its potential use in non-invasive cancer diagnostics.Abstract Background/Objectives: Liquid biopsy using cfDNA has emerged as a promising, minimally invasive alternative to traditional tissue biopsy for detecting cancer-associated mutations. However, the extremely low proportion of mutant DNA in cfDNA poses a major challenge for accurate detection, especially when using conventional sequencing methods. To address this limitation, we sought to develop a highly sensitive diagnostic strategy to selectively enrich rare mutant sequences and improve the detection of clinically important mutations in patients with NSCLC. Methods: We established a CRISPR/Cas12a-based diagnostic system designed to selectively cleave WT DNA, thereby increasing the relative abundance of mutant DNA in cfDNA samples. Following Cas12a-mediated WT cleavage, the remaining DNA was subjected to PCR amplification for mutation identification. The system was applied to plasma cfDNA from blood samples of 48 NSCLC patients to evaluate its ability to detect two major EGFR mutations: L858R and exon 19 deletion. Results: The CRISPR/Cas12a-based diagnostic system effectively identified low-frequency EGFR mutations in cfDNA. Specifically, all 7 L858R-positive samples and 6 out of 11 samples harboring exon 19 deletions-previously validated through tissue biopsy-were successfully detected. This demonstrated a high degree of concordance between our liquid biopsy approach and conventional diagnostic methods. Conclusions: Our findings highlight the potential of the CRISPR/Cas12a-based mutation enrichment system as a powerful tool for detecting rare oncogenic mutations in liquid biopsy samples. This technique enhances diagnostic sensitivity and could be broadly applicable for the non-invasive detection of various genetic alterations in cancer and other diseases.

키워드