美国麻省总医院Hakho Lee等研究人员合作使用SCOPE实现细胞外囊泡mRNA的突变分析。该项研究成果于2024年10月7日在线发表在《自然—生物技术》杂志上。

研究人员表示，液体活检中细胞外囊泡（EV）中的信使RNA（mRNA）测序可以提供临床信息，如体细胞突变、耐药性谱和肿瘤复发。尽管如此，由于其在液体活检中的低丰度，EV mRNA仍未得到充分利用，且需要大样本体积或专门的分析技术。

研究人员介绍了一种用于EV mRNA检测的平台——自放大和CRISPR辅助操作以分析EV（SCOPE）。SCOPE利用CRISPR介导的目标RNA识别，通过Cas13启动复制和信号放大，达到亚阿摩尔检测限，同时保持单核苷酸分辨率。

作为概念验证，研究人员设计了针对KRAS、BRAF、EGFR和IDH1基因关键突变的探针，优化了单锅检测的协议，并实现了多样本检测的自动化设备。研究人员验证了SCOPE在动物模型中检测早期肺癌的能力，监测了结直肠癌患者的肿瘤突变负担，并对胶质母细胞瘤患者进行了分层。SCOPE可以加快结果读取，增强EV在精准肿瘤学中的临床应用。

附：英文原文

Title: Amplifying mutational profiling of extracellular vesicle mRNA with SCOPE

Author: Song, Jayeon, Cho, Mi Hyeon, Cho, Hayoung, Song, Younseong, Lee, Sung Woon, Nam, Ho Chul, Yoon, Tae Ho, Shin, Jong Cheol, Hong, Jae-Sang, Kim, Yejin, Ekanayake, Emil, Jeon, Jueun, You, Dong Gil, Im, Sung Gap, Choi, Gyu-Seog, Park, Jun Seok, Carter, Bob C., Balaj, Leonora, Seo, An Na, Miller, Miles A., Park, Soo Yeun, Kang, Taejoon, Castro, Cesar M., Lee, Hakho

Issue&Volume: 2024-10-07

Abstract: Sequencing of messenger RNA (mRNA) found in extracellular vesicles (EVs) in liquid biopsies can provide clinical information such as somatic mutations, resistance profiles and tumor recurrence. Despite this, EV mRNA remains underused due to its low abundance in liquid biopsies, and large sample volumes or specialized techniques for analysis are required. Here we introduce Self-amplified and CRISPR-aided Operation to Profile EVs (SCOPE), a platform for EV mRNA detection. SCOPE leverages CRISPR-mediated recognition of target RNA using Cas13 to initiate replication and signal amplification, achieving a sub-attomolar detection limit while maintaining single-nucleotide resolution. As a proof of concept, we designed probes for key mutations in KRAS, BRAF, EGFR and IDH1 genes, optimized protocols for single-pot assays and implemented an automated device for multi-sample detection. We validated SCOPE’s ability to detect early-stage lung cancer in animal models, monitored tumor mutational burden in patients with colorectal cancer and stratified patients with glioblastoma. SCOPE can expedite readouts, augmenting the clinical use of EVs in precision oncology.

DOI: 10.1038/s41587-024-02426-6

Source: https://www.nature.com/articles/s41587-024-02426-6