Amplifying mutational profiling of extracellular vesicle mRNA with SCOPE

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.

Extended Data Fig. 1 |. Stratifying glioma patients.

(a) SCOPE analyzed plasma samples collected from radiologically confirmed glioma patients. Tissue samples were used for clinical pathology. (b) Target RNA sequences for glioma-SCOPE analyses. IDH1 probes were designed to detect wild-type (WT) and single nucleotide mutation (R132H), and EGFR probes to detect WT as well as the variant III (EGFRvIII) resulting from genomic deletion of exons 2–7 in the EGFR gene. (c) The specificity of glioma-SCOPE probes was evaluated. The designed probes achieved a high signal contrast (>30) between on-target and off-target samples. Synthetic RNA samples (1 nM) were used. The heatmap displays mean values from technical triplicate measurements. a.u., arbitrary unit. (d) Application of SCOPE to profile EVs for IDH1 (WT, R132H) and EGFR (WT, vIII). EVs were harvested from glioma cell lines. The results confirmed that EVs reflected the genotype of parent glioma cells. Data are shown as mean ± s.d. from biological triplicates. a.u., arbitrary unit. (e) Plasma EVs were analyzed via SCOPE for IDH1 (WT, R132H) and EGFR (WT, vIII). Control samples were from healthy donors (n = 15). Glioma samples were from patients with different genotypes: EGFR amplification (GBM-WT; n = 20), IDH1-R132H mutation (n = 20), and EGFRvIII mutation (n = 20). The SCOPE assay identified glioma patients and correctly stratified them according to their molecular genotypes. The heatmap shows mean values from technical triplicate measurements.

Figure 1.. Self-amplified and CRISPR-aided operation to profile extracellular vesicles (SCOPE).

(a) SCOPE mechanism. The assay couples Cas13a/crRNA and polymerase reactions through a signal template (bottom). The signal template contains an RNA segment with a quenched fluorescent dye and a DNA template for T7 polymerase. The fluorescent signal is initially quenched (F, fluorescent dye; Q, quencher). (b) Assay flow. (i) Cas13a/crRNA first recognizes the EV RNA target, which activates its ribonuclease function. (ii) The activated enzyme cuts the RNA segment in the signal template, generating a fluorescent signal and exposing the DNA template. (iii) T7 RNA polymerase binds to the promoter region of the DNA template and synthesizes target RNA replicas. (iv) The amplified target replicas bind to free Cas13a/crRNA, enforcing the overall assay process. Note that the initial CRISPR-mediated target recognition is necessary to unlock RNA replication and signal generation. (c) SCOPE workflow for onsite diagnostics. EVs are isolated from clinical samples and lysed. EV lysates are then placed in surface-treated tubes for RNA extraction (10 min). Subsequently, the SCOPE reaction is performed in a compact, portable device (30 min). The assay produces molecular information within our hour, enabling same-day clinical decisions.

Figure 2.. SCOPE device engineering.

(a) Single-tube system for SCOPE assay. We used PCR tubes coated with poly[(2-dimethylamino)ethyl acrylate] (pDMAEA) polymer. With EV lysates added, the dissolved polymer was positively charged initially, forming polyplexes with negatively charged nucleic acids. Following brief centrifugation to collect polyplexes, we then added SCOPE reagents. The polymer underwent charge-shifting hydrolysis to release nucleic acids, and the SCOPE reaction continued in the same tube. (b) Comparison of RNA extraction yield between pDMAEA tubes and standard silica-based columns. The pDMAEA method demonstrated similar extraction yields as the column devices, with an average extraction yield of 96% across different input RNA amounts (3, 30, 60, and 100 ng). Extracting nucleic acids with pDMAEA tubes was fast (10 min) and compatible with SCOPE reactions. Data are shown as mean ± s.d. from technical triplicates. (c) Integrated device for onsite SCOPE tests. This compact device accommodated 16 samples in a tray-like sample holder. The tray was wrapped with a flexible heater and docked to the mainframe that regulated the heater. Fluorescent signals were detected by an optical module (top right). The system was connected to a tablet via wireless communication. A user-friendly graphical interface (lower right) in a tablet provided intuitive windows for system operation and data analysis. (d) Upon completion of the reaction, the optical module quantified fluorescent signals emitted from the samples. A single module linearly scanned the sample tray, simplifying the system design. (e) The SCOPE device was used to process samples containing different amounts of synthetic CD63 RNA. The SCOPE results were highly consistent for a given RNA concentration, regardless of the sample location in the heating block. The coefficient of variation was <1.5%. The bar represents a mean fluorescent signal from four different sites in the heating block. Color codes matched the sample location with RNA concentration. a.u., arbitrary unit.

Figure 3.. SCOPE assay kinetics.

(a) SCOPE integrates two enzymatic reactions via the signal template: i) Cas13a/crRNA generates fluorescence by degrading the RNA segment in the signal template, and ii) T7 polymerase replicates RNA targets. SCOPE reactions commence upon initial recognition of mRNA targets by Cas13a/crRNA. (b) When performed independently, Cas13a/crRNA and T7 reactions exhibited a linear increase in end product over time (left panels). Coupling these two reactions enabled SCOPE to emulate first-order rate kinetics. The analytical signals amplified exponentially, reaching a steady state within 30 min (right panels). Data are shown as mean ± s.d. from technical triplicates. (c) Assay validation. Signal intensity was maximized when all SCOPE assay components were present (red bar). Removing T7 polymerase diminished the signal due to the absence of target RNA amplification (blue bar). Assay conditions that hindered the initial mRNA recognition produced negligible signals (grey bars). The initial target RNA (KRAS^G12D) concentration was fixed at 1 nM. Data are shown as mean ± s.d. from technical triplicates. a.u., arbitrary unit.

Figure 4.. SCOPE assay characterization.

(a) SCOPE (30 min) and conventional RT-PCR (80 min) were employed to analyze samples containing varying concentrations of synthetic KRAS^G12D RNA. SCOPE exhibited a detection limit (LOD) of 8.5 copies/ μL (14.2 zM), outperforming RT-PCR (LOD of 1.1 × 10¹⁰ copies/ μL, 18.6 pM). Data are displayed as mean ± s.d. from technical triplicates. Some error bars are too small to be visible. The net intensity ( ΔIntensity) was obtained by removing background fluorescent signals (no-target controls). a.u., arbitrary unit. (b) EV samples were prepared from KP1.9 cell culture and analyzed for KRAS^G12D mRNA. SCOPE’s LOD was approximately 10⁵ EVs/mL, whereas RT-PCR’s LOD was about 10⁸ EVs/mL. In addition, SCOPE displayed a broader dynamic range than RT-PCR. Data are shown as mean ± s.d. from technical triplicates. (c) The specificity of SCOPE probes targeting KRAS wild type (WT) and its mutated subtypes (G12C, G12D, G12S, G12V) was evaluated. SCOPE displayed high signal contrast between on-target and off-target samples. At a target concentration of 1 nM, the contrast ratio exceeded 30. The heatmap shows mean values from technical triplicate measurements. (d) A mixture of KRAS^WT and KRAS^G12D synthetic RNA samples was prepared, varying the variant allele fractions (VAFs). SCOPE detected KRAS^G12D down to 0.01% VAF, with a signal significantly different from the background (p = 0.011; unpaired, two-sided t-test). The bars represent mean values from technical triplicate measurements.

Figure 5.. Early cancer detection with SCOPE.

(a) Experimental design. A genetically engineered mouse model was used to mimic the development of non-small cell lung cancer. The animal had a Cre-activatable Kras^LSL-G12D/+; Trp53^flox/flox genetic background (KP model). Tumor growth was initiated through intranasal administration of adenoviruses expressing Cre recombinase (Ad-Cre). Serial blood samples were collected from the animals before tumor induction and at weekly intervals after that. (b) In vitro validation of SCOPE for KRAS genotyping. Cell lines with distinct KRAS genetic profiles were used: H2228 (WT/WT), KP1.9 (G12D/WT), and A549 (G12S/G12S). EVs were isolated from cell culture media. SCOPE KRAS assays on these EV samples yielded results consistent with the cellular KRAS status. Data are presented as mean ± s.d. from biological triplicates. a.u., arbitrary unit. (c) Tumor development in the Cre-treated mice was corroborated via immunohistochemistry on lung tissue specimens. Lesions positive for Kras^G12D protein were detectable one week after tumor induction, and the lesions enlarged in subsequent weeks. Staining was performed two different samples, and representative images are shown. (d) SCOPE analyses on EV Kras^G12D mRNA during tumor development. Serial blood samples were collected over eight weeks from the same individual mice. In the non-cancer cohort (n = 3), the SCOPE signal remained at background values throughout the duration. In the tumor-bearing cohort (n = 10), the SCOPE signal increased from background values one week after tumor induction and reached saturation approximately three weeks after the onset of tumor growth. The heatmap shows mean values from technical triplicate measurements. (e) Estimation of Kras^G12D mRNA copy numbers. SCOPE results in (d) were converted based on the titration curve (Fig. 4a). In the tumor-bearing cohort, the copy number was below the baseline (< 1) initially and then significantly increased (p = 0.042, paired two-sided t-test) one week after tumor induction. Each data point is a mean value from technical triplicate measurements.

Figure 6.. Monitoring colorectal cancer patients.

(a) Plasma samples were collected from colorectal cancer (CRC) patients before and after surgery, as well as during standard care. Tissue samples were obtained during surgery and analyzed for the KRAS gene. (b) Plasma EVs were analyzed via SCOPE for a panel of KRAS mRNA mutations. Samples were from non-CRC controls (n = 15) and pre-surgery CRC patients (n = 107). SCOPE results reflected KRAS mutation status from tumor tissue analysis. KRAS signals from EVs were normalized against that of GAPDH. The heatmap shows mean values from technical triplicates. (c) Images of tissue staining (immunohistochemistry). For a patient positive with KRAS^G12D EV mRNA, the tumor tissue was also positively stained for KRAS^G12D protein. The staining experiments were performed on two different tissues, and representative images are shown. Scale bar, 20 μm. (d) The results from (b) were converted into the ratio (R_KRAS = KRAS^MT/KRAS^WT) of SCOPE signals between specific KRAS mutation (KRAS^MT) and KRAS wild type (KRAS^WT). The threshold for KRAS^MT positivity was established using profiling data from non-CRC controls and KRAS^WT CRC patients. For each specific KRAS mutation, the R_KRAS values of CRC patients harboring that particular mutation exceeded those of non-CRC controls and KRAS^WT CRC patients. Half-filled circles (◒) indicate patients whose KRAS mutation type is concordant between SCOPE and clinical tissue analyses. Each data point is a mean value from technical triplicates. (e) Seventeen KRAS^MT CRC patients were followed up for one year. For all patients, R_KRAS values immediately after surgery were lower than their initial values. Among the non-recurrent patients (n = 14), R_KRAS decreased below the threshold (5.5%). In the recurrent patients (n = 3), R_KRAS values eventually returned to pre-surgery levels. Each data point is a mean value from technical triplicates. (f) In the non-recurrent CRC patients, the R_KRAS values would fall below the threshold approximately ten days following surgery. The dotted line is the optimal fit for the post-surgery data, R_KRAS ~ d^0.3, where d is the number of days after surgery. The shaded region represents the 95% confidence interval. Data are shown as mean ± s.d. from 14 patients.