Researchers are improving the efficiency and effectiveness of CRISPR Diagnostic Technology
Changchun Liu, professor of biomedical engineering at UConn Health, has developed a new method that improves existing diagnostic technology for a faster, sensitive and implementable approach to molecular diagnostics.
The research, published in Nature communication, unravels new potential for simple and sensitive nucleic acid detection in various diagnostic settings—including early cancer diagnosis and infectious disease detection—using new and improved clustered regularly interspaced short palindromic repeats (CRISPR) technology. CRISPR technology uses an enzyme that can be programmed to seek out a specific stretch of nucleic acid and intervene in it.
PCR-based nucleic acid detection methods have long been considered the gold standard due to their high sensitivity and specificity. However, there is a growing need for alternative solutions that are fast, cost-effective and easy to use. CRISPR technology is emerging as a powerful tool for nucleic acid detection due to its affordability and simplicity.
The existing CRISPR method requires two steps – a pre-amplification step of target nucleic acids and a detection of CRISPR enzymes. Although this method is simple and efficient than standard PCR-based methods, the two-step requirement and the need for a separate pre-amplification step limit practical application and lack quantitative detection capability.
“To meet stringent sensitivity specifications in clinical tests, CRISPR-based nucleic acid detection typically requires target nucleic acid pre-amplification,” says Changchun Liu. “Recently, we have revealed an asymmetric trans-cleavage behavior of competing crRNAs in the CRISPR-Cas12a reaction. Based on this finding, we developed a sensitive, amplification-free, asymmetric CRISPR assay for the quantitative detection of nucleic acids.”
Using CRISPR-Cas12a, an RNA-directed DNA enzyme that can be exploited for gene editing, the researchers developed a new CRISPR assay by exploiting a unique, competitive reaction between a full-length crRNA and a split crRNA. The researchers found that the reaction can induce signal amplification, which can significantly improve the target detection signal – increasing the diagnostic tool’s sensitivity. In addition, the researchers found that CRISPR-Cas12a can recognize fragmented RNA/DNA targets, allowing researchers to detect microRNA quantitatively without the need for the pre-amplification step.
This improved diagnostic technology, without the need for pre-amplification, achieved an attomolar detection sensitivity, 1,000 times more sensitive than the conventional CRISPR detection.
To test this new technology for efficacy in a clinical setting, the researchers used the new CRISPR assay to analyze and quantify microRNA-19a biomarker in plasma samples from bladder cancer patients, successfully demonstrating the potential of this new CRISPR technology as a powerful tool for simple , fast and sensitive liquid biopsy.
“Liquid biopsy is just one example of the clinical applications of our asymmetric assay. We envision that it may have broad clinical implications in early cancer diagnosis and infectious disease detection,” said Jeong Moon, a postdoctoral researcher in the Liu lab and first author of the paper.
Liu is a prolific inventor, and UConn’s Technology Commercialization Services (TCS) has filed a portfolio of patents on novel molecular diagnostic technologies developed in Liu’s laboratory.
Ana Fidantsef, Industry Liaison at TCS says, “It is very exciting that this particular amplification-free, CRISPR-Cas12a molecular diagnostic test achieves attomole-level detection sensitivity that has never been achieved before using CRISPR-Cas12a technology. We are eager to reach to industry and transfer this technology to commercialization”.
An overview of the technology can be found here.
This work was supported in part by NIH U01CA269147 and U01AI148306.
