Cancer Diagnosis Made Easier with CRISPR Based Tool

A team of University of Florida researchers has created a potential new CRISPR-powered technology for non-invasive blood tests that could aid clinicians in the early cancer diagnosis.

The researchers proved that their technique is as successful as the widely used reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays for cancer diagnosis and that it can be used in conjunction with a simple portable device for point-of-care clinical testing.
The study was headed by Yong Zeng, Ph.D., an associate professor in the department of chemistry, an affiliate faculty member in the J. Crayton Pruitt Family department of biomedical engineering, and a member of the UF Health Cancer Center. The samples evaluated in the study were collected in collaboration with the department of surgery at the University of Florida College of Medicine.

The approach detects microRNAs, which are small RNA molecules involved in gene expression regulation, in extracellular vesicles, which are tiny particles circulating in the blood.

MicroRNAs have emerged as intriguing candidates for generating cancer biomarkers in human fluids such as blood. However, clinical applicability has been limited due to the complexity of microRNAs and the lack of a sensitive enough technique to detect them.

Extracellular vesicles are nanosized particles, actively released by cells, that play important roles in cell functions and diseases by shuttling biomolecules among cells. Tumor cells seem to more aggressively produce the extracellular vesicles in microRNAs associated with disease, “making them an exciting source for exploring new cancer markers,” Zeng said.

CRISPR technology, a powerful gene editing technique that was the subject of the 2020 Nobel Prize in Chemistry, has recently sparked attention as a means of developing new bioassays for disease diagnosis. However, previous CRISPR assays required manual handling and were not as sensitive as the gold standard, RT-qPCR.

“Our idea was to design a way to simplify the entire workflow into ‘one pot,'” Zeng said. “We designed a fast, sensitive method to detect microRNAs that is simpler and has a low risk of cross-contamination.”

The “one pot” method requires that every chemical agent required, except the sample, be stored in a single test tube. To conduct the study, only the microRNA sample is required to induce a reaction.

“To our surprise, we found that our method has comparable sensitivity to PCR tests,” Zeng said. “Our method has excellent analytical and diagnostic performance while substantially expediting the workflow.”

Given the disease’s high death rate, the researchers concentrated on adopting the technology for pancreatic cancer. Early detection of pancreatic cancer is challenging. Often, by the time a tumor is detected, it has spread and cannot be entirely removed, eliminating the possibility of a cure.

“Compared to other types of cancers, diagnostic testing for pancreatic cancer has not changed significantly in nearly 50 years,” Zeng said. “There is significant interest in developing biomarkers for pancreatic cancer diagnosis.”

The technology, dubbed “EXTRA-CRISPR,” is a game changer in the field of microRNA testing, according to He Yan, Ph.D., a postdoctoral researcher in Zeng’s group and the paper’s first author.

“Our method is very promising for diagnosis of cancer, such as pancreatic cancer, when combined with robust microRNA biomarkers and for point-of-care testing,” he said. “In the future, this method can be coupled with a very simple, low-cost portable device to make pancreatic cancer detection simpler but still reliable.”

The team demonstrated as a proof of concept that the new one-pot assay could be adapted for two detection modalities routinely used for point-of-care testing. Initially, a portable smartphone-based gadget was prototyped utilizing off-the-shelf components such as a blue LED illuminator, a plastic filter, and a coffee mug warmer. These components were integrated on a 3D-printed body portion by the team. To measure the target markers, a smartphone was put on the gadget to capture fluorescence photos of the test vials following the reaction.

In addition to the fluorescence detector, they built an instrument-free point-of-care device by combining the EXTRA-CRISPR assay with a commercially available lateral-flow test strip. The researchers investigated both point-of-care approaches for detecting pancreatic cancer, and the results were consistent with those obtained using a benchtop PCR equipment.

Based on their findings, the researchers have filed a patent application to help make the one-pot CRISPR assay and point-of-care technology widely available for fundamental research and clinical applications.

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