Closer look into EV mRNA through SCOPE

Extracellular vesicles (EVs) have become important targets for medical testing. These tiny particles, released by cells, carry various molecules that can tell us about the cells they came from. Detecting genetic material, particularly mRNA in EVs, can provide valuable medical information, such as tumor burden, specific genetic changes, and treatment responses. However, detecting EV mRNA is challenging because EVs contain very small amounts. Current analytical methods often require large blood samples (over 2 mL) and advanced equipment.

In a paper published in Nature Biotechnology, CSB researchers report a breakthrough inspired by CRISPR technology. Unlike other CRISPR-based tests, this new method uses CRISPR to amplify both target mRNA and fluorescent signals. The approach is based on a specially designed hybrid molecule that combines a fluorescent reporter probe with a DNA template for mRNA replication – a CRISPR protein (Cas13a) first recognizes specific mRNA targets, which then triggers the copying of more mRNA and increases of the detection signal. This unique mechanism enables the assay to discriminate between single-base differences while detecting extremely small amounts of mRNA (a few hundred copies in one-milliliter solution).

This new test, called SCOPE (Self-amplified and CRISPR-aided Operation to Profile EVs), was validated through pre-clinical and clinical studies. In one notable example, SCOPE effectively differentiated normal KRAS mRNA in EVs from its mutated subtypes, performing better than a commercial mutation test. This ability was crucial in developing an EV test to detect early-stage lung cancer in animal models, monitor genetic changes in colorectal cancer patients during standard treatment, and classify brain cancer patients. This technique could be adapted to detect other molecules (like proteins or microRNAs), making EVs even more useful for liquid biopsy. It could also be a powerful tool in different fields, such as detecting infectious diseases or in agriculture, where quick, sensitive, and specific molecular tests are needed.

A general overview and background story are available in the Research Briefing accompanying the paper. The current work complements another CRISPR-based technology, CreDiT (CRISPR Enhanced Digital Testing), which the researchers developed for DNA detection.