CRISPR/Cas13a-Based MicroRNA Detection in Tumor-Derived Extracellular Vesicles

Area of Science:

• Biochemistry
• Molecular Biology
• Nanotechnology

Background:

• MicroRNAs (miRNAs) within extracellular vesicles (EVs) are crucial in cancer development.
• Accurate quantification of EV miRNAs is vital for cancer diagnostics and monitoring.
• Existing PCR-based methods for EV miRNA analysis are complex and provide only bulk measurements.

Purpose of the Study:

• To develop an amplification-free and extraction-free method for quantifying specific miRNAs in intact EVs.
• To utilize a CRISPR/Cas13a sensing system encapsulated within liposomes for EV miRNA detection.
• To enable multiplexed analysis of protein and RNA markers in single EVs.

Main Methods:

• Liposomes containing CRISPR/Cas13a components were fused with EVs.
• Specific miRNA-positive EV counts were quantified using the CRISPR/Cas13a system.
• Multiplexed analysis involved capturing EpCAM-positive EVs and quantifying miR-21-5p in subpopulations.

Main Results:

• The method accurately quantifies specific miRNA-positive EV counts (e.g., miR-21-5p) in ovarian cancer EVs (2%-10%) compared to benign cells (<0.65%).
• Results showed excellent correlation with the gold-standard RT-qPCR method.
• Elevated miR-21-5p levels were detected in EVs from cancer patients' plasma compared to healthy controls.

Conclusions:

• The developed CRISPR/Cas13a system offers a direct, sensitive method for detecting specific miRNAs in intact EVs without RNA extraction.
• This approach facilitates accurate quantification of EV miRNAs for cancer diagnosis and monitoring.
• The system opens possibilities for multiplexed single EV analysis, combining protein and RNA marker detection.