Rapid in situ mutation detection in extracellular vesicle DNA

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Oncology

Background:

• Traditional cancer mutation detection assays often require PCR and sequencing, which are time-consuming and rely on specialized equipment.
• The COVID-19 pandemic highlighted challenges in cancer mutation analysis due to high demand for viral testing, impacting patients on targeted therapies.
• There is a need for rapid, accessible, and equipment-independent methods for cancer mutation detection.

Purpose of the Study:

• To develop a rapid, PCR- and sequencing-free DNA mutation detection assay using CRISPR-Cas12a technology.
• To enable efficient detection of cancer-driving mutations in extracellular vesicles (EVs) for improved cancer diagnosis and monitoring.
• To create a user-friendly assay compatible with fundamental laboratory equipment like microplate readers.

Main Methods:

• Utilized CRISPR-Cas12a complexes and fluorescence-quenching (FQ) probes encapsulated within liposomes.
• Introduced Cas12a and FQ probes into tumor-derived extracellular vesicles (EVs) via membrane fusion.
• Activated Cas12a, upon hybridizing with target DNA, induces trans-cleavage of FQ probes, generating a quantifiable fluorescence signal.

Main Results:

• Successfully detected the EGFR L858R mutation in EV DNA within 30 minutes, eliminating the need for laborious DNA extraction and purification.
• Achieved a sensitivity of 86.7%, specificity of 90%, and accuracy of 87.5% in a cohort of 30 advanced non-small cell lung cancer (NSCLC) patients and 10 healthy donors.
• Demonstrated a limit of detection of approximately 8 × 10^5 EVs, corresponding to a mutation allele frequency (MAF) of ~10%, suitable for detecting mutations in advanced lung cancer without target amplification.

Conclusions:

• The developed Cas12a-based assay provides a rapid and efficient method for DNA mutation detection in EVs.
• This assay eliminates the need for PCR, sequencing, and extensive sample preparation, making it accessible for routine diagnostics.
• The technology holds promise for future advancements in multiplex and high-throughput mutation detection, facilitating at-home self-diagnosis and treatment monitoring.