Tsou Jen-Hui, Leng Qixin, Jiang Feng
Department of Pathology, University of Maryland School of Medicine, 10 S. Pine St. Baltimore, MD 21201, USA.
Diagnostics (Basel). 2020 Feb 19;10(2):114. doi: 10.3390/diagnostics10020114.
The detection of EGFR mutations in circulating cell-free DNA can enable personalized therapy for cancer. The current techniques for detecting circulating EGFR mutations are expensive and time-consuming with moderate sensitivity. Emerging CRISPR is revolutionizing medical diagnostics and showing a great promise for nucleic acid detection. This study aims to develop CRISPR-Cas12a as a simple test to sensitively detect circulating EGFR mutations in plasma. Serially diluted samples of DNA containing heterozygous EGFR mutations (L858R and T790M) in wild-type genomic DNA are concurrently tested for the mutations by a CRISPR-Cas12a system and droplet digital PCR (ddPCR). The CRISPR-Cas12a system can detect both L858R and T790M with a limit of detection of 0.005% in less than three hours. ddPCR detects the mutations with a limit of detection of 0.05% for more than five hours. Plasma samples of 28 lung cancer patients and 20 cancer-free individuals are tested for the EGFR mutations by CRISPR-Cas12a system and ddPCR. The CRISPR-Cas12a system could detect L858R in plasma of two lung cancer patients whose tissue biopsies are positive for L858R, and one plasma sample of three lung cancer patients whose tissue biopsies are positive for T790M. ddPCR detects L858R in the same two plasm samples, however, does not detect T790M in any of the plasma samples. This proof of principle study demonstrates that the CRISPR-Cas12a system could rapidly and sensitively detect circulating EGFR mutations, and thus, has potential prognostic or therapeutic implications.
检测循环游离DNA中的表皮生长因子受体（EGFR）突变可实现癌症的个性化治疗。目前检测循环EGFR突变的技术昂贵且耗时，灵敏度中等。新兴的CRISPR正在彻底改变医学诊断，并在核酸检测方面展现出巨大潜力。本研究旨在开发一种基于CRISPR-Cas12a的简单检测方法，用于灵敏检测血浆中循环的EGFR突变。通过CRISPR-Cas12a系统和液滴数字PCR（ddPCR）同时检测野生型基因组DNA中含有杂合EGFR突变（L858R和T790M）的系列稀释DNA样本中的突变。CRISPR-Cas12a系统可在不到三小时内检测到L858R和T790M，检测限为0.005%。ddPCR检测这些突变的检测限为0.05%，耗时超过五小时。通过CRISPR-Cas12a系统和ddPCR对28例肺癌患者和20例无癌个体的血浆样本进行EGFR突变检测。CRISPR-Cas12a系统能够在两名组织活检L858R呈阳性的肺癌患者血浆中检测到L858R，在三名组织活检T790M呈阳性的肺癌患者的一份血浆样本中检测到T790M。ddPCR在相同的两份血浆样本中检测到了L858R，但在任何血浆样本中均未检测到T790M。这项原理验证研究表明，CRISPR-Cas12a系统能够快速、灵敏地检测循环EGFR突变，因此具有潜在的预后或治疗意义。
