文章：

CRISPR结合双错配引导RNA增强肺癌患者循环游离DNA中低频单碱基EGFR突变检测灵敏度

CRISPR with a Double Mismatch Guide RNA Enhances Detection Sensitivity for Low-Frequency Single-Base EGFR Mutation in Circulating Cell-Free DNA of Lung Cancer Patients

原文发布日期：16 October 2025

DOI: 10.3390/cancers17203343

类型: Article

开放获取: 是

英文摘要：

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.

摘要翻译： 

背景/目的：利用循环游离DNA（cfDNA）进行液体活检已成为一种有前景的微创检测方法，可作为传统组织活检的替代方案用于检测癌症相关突变。然而，cfDNA中突变DNA比例极低，对准确检测构成重大挑战，尤其是在使用常规测序方法时。为突破这一局限，我们致力于开发一种高灵敏度诊断策略，以选择性富集稀有突变序列，提升非小细胞肺癌（NSCLC）患者临床重要突变的检出能力。方法：我们构建了一种基于CRISPR/Cas12a的诊断系统，该系统通过特异性切割野生型DNA，从而提高cfDNA样本中突变DNA的相对丰度。经Cas12a介导的野生型DNA切割后，对剩余DNA进行PCR扩增以鉴定突变。我们将该系统应用于48例NSCLC患者血浆cfDNA样本，评估其检测两种主要EGFR突变（L858R突变和19号外显子缺失）的能力。结果：基于CRISPR/Cas12a的诊断系统能有效识别cfDNA中的低频EGFR突变。具体而言，所有7例L858R阳性样本及11例携带19号外显子缺失样本中的6例（均经组织活检验证）均被成功检出，表明本液体活检方法与常规诊断手段具有高度一致性。结论：本研究证实基于CRISPR/Cas12a的突变富集系统可作为检测液体活检样本中罕见致癌突变的有效工具。该技术显著提升了诊断灵敏度，有望广泛应用于癌症及其他疾病多种遗传变异的无创检测领域。

原文链接：

CRISPR with a Double Mismatch Guide RNA Enhances Detection Sensitivity for Low-Frequency Single-Base EGFR Mutation in Circulating Cell-Free DNA of Lung Cancer Patients