by Michael D. O’Neill, Editor & Publisher, BioQuick News
Researchers at Mass General Hospital, Harvard Medical School, and collaborating institutions in the Republic of Korea have developed a CRISPR-based method (SCOPE) to detect mRNA at sub-attomolar levels in extracellular vesicles (EVs), while maintaining single-nucleotide resolution. SCOPE (self-amplified and CRISPR-aided operation to profile EVs) leverages CRISPR-mediated recognition of target RNA using CRISPR-associated protein 13 (Cas13) to initiate replication and signal amplification. As a proof of concept, researchers 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. They also 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.
The new work is described in an October 7, 2024 article in Nature Biotechnology that is titled “Amplifying Mutational Profiling of Extracellular Vesicle mRNA with SCOPE.” The article is accompanied by a brief summary written by two of the article’s corresponding authors (Cesar M. Castro and Hakho Lee of Mass General/Harvard Medical School). This summary is titled “A CRISPR-Based Method for Detecting mRNA in Extracellular Vesicles.”
The summary includes an expert opinion on the work by An Hendrix of Ghent University in Belgium: “The SCOPE assay holds the potential to advance the analysis of scarce amounts of mRNAs in biospecimens, including mRNA encapsulated in EVs. The authors have characterized the EV samples in compliance with MISEV guidelines [Minimal Information for Studies of Extracellular Vesicles], enzymatic treatments have been used to confirm that the analyzed mRNA is encapsulated in EVs, and diverse preparation methods have been implemented to explore their compatibility with the SCOPE assay.”
In addition, there is a comment from the Nature Biotechnology editorial team: “In this manuscript, Song and colleagues introduce and evaluate the SCOPE assay, a novel integrated platform for the detection of EV-associated mRNA in biospecimens utilizing CRISPR-mediated target recognition, replication, and signal amplification. The SCOPE assay employs a sophisticated Cas13/T7 polymerase-mediated dual target amplification loop, alongside Cas13-driven release of fluorescent dye from DNA/RNA signal sequences.”
Liquid Biopsy
Liquid biopsy, where tumor biomarkers are detected noninvasively from body fluids (blood, plasma, saliva, spinal fluid), has been pursued in order to offer diagnosis, prognosis, and recurrence monitoring of cancer. Molecular targets that have been looked at include tumor-derived cell-free DNA, microRNA, and messenger RNA. Limitations of cell-free DNA include its low concentration and the fact that it primarily represents dying cells. In contrast, according to the summary authors, the detection of messenger RNA (mRNA) offers potential advantages as it is more abundant than circulating tumor DNA and could provide insights into the functional states of tumors, potentially benefiting preclinical and clinical investigations.
EVs and mRNA
The summary authors note that EVs have emerged as promising targets for mRNA analysis as they protect mRNA from enzymatic degradation and they might serve as cellular surrogates reflecting the properties of the parent cell. However, the detection of EV-mRNA has faced technical challenges. EVs typically contain low copy numbers of mRNA molecules and tumor-derived EVs constitute only a minor fraction of the total EVs in blood. Consequently, EV-mRNA analyses currently require large sample volumes (>2 mL plasma) and advanced instrumentation.
New Work
The team behind the newly described work reports the development of a CRISPR-based assay that both recognizes and amplifies target EV-mRNA, achieving sub-attomolar sensitivity with single nucleotide resolution. The method enables the detection of low-abundance mRNA in EVs, providing clinically relevant information for precision oncology.
Summary Description
In their summary, corresponding authors Castro and Lee write “We drew inspiration from the CRISPR (clustered regularly interspaced short palindromic repeats) approach to biosensing. CRISPR technologies are increasingly adopted for molecular diagnostics because of their sequence-specific nuclease activity. CRISPR-associated (Cas) proteins become active nucleases upon recognizing target nucleic acids. This property is used to cut quenched fluorescent reporter probes; these Cas-generated signals can be detected and usually increase linearly over time. Most assays amplify target nucleic acids before Cas recognition, but any errors during this amplification could lead to false positives.”
“We repurposed Cas activity to amplify both the target mRNA and the detection signal. The approach employs a signal template, a hybrid design that integrates a fluorescent reporter probe with a template for mRNA amplification. Unlike other approaches, the assay requires Cas13a to recognize its specific mRNA target before RNA replication and the exponential amplification of fluorescence signals is triggered. This distinct mechanism enables the assay to discriminate between single-nucleotide polymorphisms (SNPs) expressed in EVs, a capability attributed to the high selectivity of Cas13a. Moreover, the dual amplification of mRNA and fluorescence signals enables mRNA detection down to the sub-attomolar range.”
“We dubbed this assay SCOPE (self-amplified and CRISPR-aided operation to profile EVs) and validated its performance through systematic preclinical and clinical studies. In a notable example, SCOPE effectively differentiated KRAS wild-type mRNA in EVs from the SNPs G12C, G12D, G12S, and G12V, surpassing the performance of a commercial assay for detecting these mutations. This validation of SCOPE catalyzed the design of our EV assays for monitoring tumor mutational burden in patients with colorectal cancer undergoing standard care, detecting early-stage lung cancer in mouse models, and molecularly stratifying cases of glioma.”
“We demonstrate that SCOPE detects circulating EVs harboring genetic mutations that correspond to those found in tumor tissues, further substantiating EVs as reliable liquid biopsy biomarkers. SCOPE has the potential to facilitate biomarker-driven opportunities for elucidating the mechanisms underlying cancer growth and the emergence of resistance, as well as for evaluating treatment response and identifying minimal residual disease. These outcomes could expedite clinical decision-making and the use of EVs in liquid biopsy applications.”
Possible Protein Detection in Future
The summary authors note that “Future assay development could incorporate protein detection into SCOPE’s work-flow, capitalizing on the intrinsic advantage of EVs as multi-cargo carriers. Detecting proteins would complement mRNA analyses, bridging tumor genotypes with phenotypes. For example, protein markers might provide additional insights into a tumor’s tissue of origin, information that is challenging to ascertain from mRNA assays alone. The extension of SCOPE into other fields, such as infectious diseases and agriculture, where timely, sensitive, and specific readouts are paramount, could also enhance its reach.”
[Nature Biotechnology abstract] [Nature Biotechnology summary]