Researchers at Zhengzhou University have developed what they say is an easy-to-use technology that uses CRISPR gene editing to detect small amounts of cancer-related microRNAs (miRNAs) in exosomes in plasma, and effectively distinguish between malignant and benign samples. Most cancer diagnostic techniques rely on uncomfortable and invasive procedures, such as biopsies, endoscopies, or mammograms. Blood samples could be a less unpleasant option, though only a few forms of the disease can currently be diagnosed this way.
Reporting on the simple, highly sensitive liposome-mediated membrane fusion (MFS)-CRISPR technology in ACS Sensors, Hua Gao, PhD, Kaixiang Zhang PhD, and colleagues suggest that their new technology “could have promising clinical potential for cancer diagnosis and treatment monitoring.” Their published paper describing the MFS-CRISPR platform is titled “Highly Effective Detection of Exosomal miRNAs in Plasma Using Liposome-Mediated Transfection CRISPR/Cas13a,” in which they concluded, “The proposed method is simple, fast, and sensitive, which may boost the development of minimally invasive liquid biopsy.”
Exosomes are small vesicles that pinch off from a host cell, inside of which they carry functional biomolecules, such as nucleic acids, miRNAs, mRNAs, DNAs, lipids, and proteins. So, exosomes can offer a window into the physiological state of the original cell from which they originated, because their cargos may vary under different pathological conditions, the authors noted. “Among various cargos, the aberrant miRNA expression is closely associated with multiple diseases, including cancer.”
The unique intracellular environment of cancer cells can be reflected in their exosomes through biomarkers such as miRNAs. These are very small nucleic acids, only a few nucleotides in length, that regulate protein expression in cells and can become dysregulated in tumors. Therefore, it’s possible that a blood test could someday detect cancerous cells simply by targeting these exosomal miRNAs. “Exosomal miRNAs play a critical role in cancer biology and could be potential biomarkers for cancer diagnosis,” the authors continued. However 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. “… due to the low abundance of miRNAs in the exosomes, recognizing and detecting disease-associated exosomal miRNAs in an easy-to-operate way remains a challenge,” the team stated.
Some researchers have developed systems based on the gene-editing tool CRISPR to analyze RNA and proteins in vesicles. These include a CRISPR/Case12a-based system for detecting SARS-CoV-2 RNA in extracellular vesicles, and a system for direct detection of tumor-derived extracellular vesicle proteins. But the Zhengzhou University team wanted to develop a way to detect the small numbers of cancer-related exosomal miRNAs using a different CRISPR/Cas13a platform with a unique RNase activity that was sensitive, reliable, and effective. To create the detection method, the team designed a CRISPR/Cas13a system to cut apart a fluorophore and quencher-labeled reporter molecule, then packed it into a liposome— essentially a manufactured version of an exosome. “… we used a liposome-mediated MFS to transfect CRISPR/Cas13a into exosomes, termed MFS-CRISPR …” the investigators explained. “Bacterial Cas13a possesses a unique RNase activity, and the RNA-guided trans-cleavage activity of Cas13a could cleave a fluorophore and quencher-labeled reporter, leading to enhanced fluorescence after target-RNA-triggered RNase activation.”
So, when the liposome and exosome compartments fuse 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. “The encapsulated CRISPR/Cas13a recognizes and binds to the target exosomal miRNAs and then activates trans-cleavage activity, efficiently cleaving reporters and generating amplifying fluorescence signals.”
In their reported experiments, the team targeted miRNA-21, which is involved in the development of several diseases, including breast cancer. Their results showed that the MFS-CRISPR technology could be used to successfully detect this miRNA within a mixture of similar sequences, and with high sensitivity. The team also tested the method on a group of exosomes from healthy human cells and groups derived from breast cancer cells.
These tests confirmed that the new technology consistently differentiated the cancer-related exosomes from those derived from healthy cells, showing it could be useful as a cancer diagnostic. “Directly measuring the plasma samples, the MFS-CRISPR platform is able to successfully discriminate the clinical samples between breast cancer patients and healthy donors,” they said. “According to the results of fluorescence intensity detected from clinical samples, the difference of miR-21 expression of breast cancer patients and healthy donors was significant.” The researchers suggest that their method has the potential to make cancer diagnosis and monitoring quicker and easier by analyzing blood samples.