Zhengzhou University researchers reported in ACS Sensors that they have developed a CRISPR-based method that can detect small amounts of cancer-related molecules in exosomes in plasma and effectively distinguish between malignant and benign samples.
Exosomes provide a window into the condition of the cell they originated from. Accordingly, the unique intracellular environment of cancerous cells will be reflected in their exosomes through biomarkers such as micro RNAs (miRNAs). But quantifying miRNAs has been difficult because they are present at very low levels in exosomes, requiring laborious processes that can introduce contamination and report unreliable results.
To circumvent those limitations, the Zhengzhou developed a liposome-mediated membrane fusion strategy (MFS) to transfect CRISPR/Cas13a into exosomes directly measuring exosomal miRNAs in plasma.
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To create their detection method, called MFS-CRISPR, the team designed a CRISPR/Cas13a system to cut apart a fluorophore and quencher-labeled reporter molecule, then packed it into a liposome. When the two types of compartments fused together, the CRISPR cargo would then interact with the exosomal genetic material. If the target miRNA sequence was present, the Cas13a protein became activated and cut apart the reporter molecule, producing a fluorescent signal.
In these experiments, the team targeted miRNA-21, which is involved in the development of several diseases, including breast cancer. The method successfully detected this miRNA within a mixture of similar sequences with high sensitivity. In other experiments, the researchers tested the method on a group of exosomes from healthy human cells and groups derived from breast cancer cells. The system consistently differentiated the cancer-related exosomes from those derived from healthy cells, showing it could be useful as a cancer diagnostic. The researchers say that this method has the potential to make cancer diagnosis and monitoring quicker and easier by analyzing blood samples.