(T1430-01-05) Rapid and Precise Detection of Mutations in Extracellular Vesicle DNA Using a Liposome-Based CRISPR-Cas12a System

Purpose: A mutation detection assay that does not rely on PCR or sequencing streamlines cancer diagnosis and minimizes dependence on specialized equipment. This advantage became particularly evident during the pandemic when the high demand for viral nucleic acid testing often took precedence over mutation analysis. As a result, patients undergoing targeted therapy faced significant difficulties in monitoring mutations. In this context, we present a 30-minute DNA mutation detection method using Cas12a-loaded liposomes and a microplate reader, a basic yet essential laboratory instrument.

Methods: We designed the CRISPR/Cas12a assay by incorporating specific target sequences to detect DNA mutations in extracellular vesicles (EVs) associated with lung cancer, along with dye-labeled single-strand DNA into a liposome. The CRISPR-Cas12a complex and fluorescence-quenching (FQ) probes are delivered into tumor-derived extracellular vesicles (EVs) via membrane fusion. Upon hybridization of CRISPR-RNA with the target DNA, the activated Cas12a induces trans-cleavage of FQ probes, generating fluorescence signals that enable the quantification of DNA mutations.

Results: This approach allows for the detection of the EGFR L858R mutation in EV DNA within 30 minutes, eliminating the need for labor-intensive steps such as extraction, purification, and other DNA preparation processes. It also removes the requirement for complex data analysis. In a cohort study with 10 healthy donors and 30 patients with advanced NSCLC, the assay demonstrated a sensitivity of 86.7%, specificity of 90%, and accuracy of 87.5%.

Conclusion: The detection limit of our Cas12 assay was approximately 8×10⁵ EVs, corresponding to a mutation allele frequency (MAF) of around 10%. MAF in late-stage cancers typically ranges from 5% to 50%. Thus, even without target amplification, this Cas12 assay is capable of detecting mutations in patients with advanced lung cancer. Future developments in multiplex and high-throughput mutation detection using this method could facilitate streamlined self-diagnosis and treatment monitoring at home.

Figure 1: Schematic of CRISPR-Cas12a-based EV-DNA mutation detection. EVs in plasma are immunocaptured onto anti-EpCAM antibody-grafted surfaces followed by membrane fusion with LPs containing Cas12a-crRNA and FQ probes. When crRNA hybridizes with the DNA target, activated Cas12a can cleave FQ probes, resulting in fluorescence signals. Therefore, DNA mutation can be qualitatively detected with a typical microplate reader.