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
Center for Systems Biology, Massachusetts General Hospital Research Institute, Boston, MA, USA.
Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
Nat Biotechnol. 2024 Oct 7. doi: 10.1038/s41587-024-02426-6.
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.
对液体活检中细胞外囊泡（EV）内信使核糖核酸（mRNA）进行测序，可提供诸如体细胞突变、耐药性特征及肿瘤复发等临床信息。尽管如此，由于液体活检中EV mRNA丰度较低，其仍未得到充分利用，且需要大量样本或采用专门的分析技术。在此，我们介绍一种用于EV mRNA检测的平台——用于分析EV的自扩增和CRISPR辅助操作（SCOPE）。SCOPE利用Cas13通过CRISPR介导对靶RNA的识别来启动复制和信号放大，在保持单核苷酸分辨率的同时实现了亚阿托摩尔的检测限。作为概念验证，我们针对KRAS、BRAF、EGFR和IDH1基因的关键突变设计了探针，优化了单管检测方案，并实现了用于多样本检测的自动化设备。我们验证了SCOPE在动物模型中检测早期肺癌、监测结直肠癌患者肿瘤突变负荷以及对胶质母细胞瘤患者进行分层的能力。SCOPE能够加快检测结果的得出，增强EV在精准肿瘤学中的临床应用。
