![]() "That means the system is reprogrammable, so you can change what you want to target and detect a single change in a sequence," Brogan said, adding that it may be possible to develop many variations of the reporter probe, enabling multiplexing and the detection of multiple sequences in one reaction.Īccording to Cesar Castro, a point-care-diagnostic test developer and director of the cancer program at Boston-based Mass General Hospital Cancer Center, Cas13d "is not super unique but certainly has competitive advantages" over other CRISPR enzyme subtypes including efficiency. When a pathogen is not in the sample, no cleavage occurs, signaling a negative test result.Ĭas13d has a preference for a sequence designed into the modified reporter probe, and each reporter can be designed to detect a specific genomic sequence. When a target pathogen is present in a sample, a second, modified RNA probe detects cleavage by fluorescence or by altering the color on a lateral flow strip. Like other CRISPR-Cas13 enzymes, Cas13d initially cleaves target RNA and subsequently cleaves bystander RNA in the vicinity. "CRISPR-Cas enzymes have evolved to recognize specific nucleic acid sequences, so they are highly specific to their targets," she said, adding, "They also increase the sensitivity of isothermal amplification due to their additional catalytic activity while cleaving the reporter nucleic acids." She is the first author of a paper published recently in Science Advances describing the development of a CRISPR-based, low-cost, point-of-care diagnostic that the researchers said can detect SARS-CoV-2 from unprocessed patient saliva in about one hour. MIT researcher Helena de Puig, who is not affiliated with the UCSD work, said that when Cas enzymes are combined with isothermal amplification, they add both sensitivity and specificity in infectious disease diagnostic testing. Isothermal amplification is needed so the test can reach a level of sensitivity required for SARS-CoV-2 and other infectious disease tests, but it often leads to a low level of specificity and that in turn can produce high rates of false positives, Brogan noted.Īs a result, he and his colleagues are using a CRISPR enzyme subtype, Cas13d, to compensate for a lack of specificity. The CRISPR-based platform they are developing achieves its one-hour time to result through the use of isothermal amplification, which is a fast way to amplify SARS-CoV-2 because it uses only one thermal cycle. However, he and his colleagues ultimately plan to develop inexpensive assays that could be used for testing anywhere and for many indications, including in the home. UCSD said it has inked a joint development agreement with an undisclosed diagnostic test developer with the aim of commercializing its approach, and Brogan and his colleagues recently described the development of their proof-of-concept platform in ACS Sensors, saying they obtained attomolar-level sensitivity using both fluorescence and lateral flow readouts.Īccording to Brogan, the technology may be best suited for low-resource countries without modern healthcare infrastructures, for which the test is being developed for easy deployment. If the test and its platform can be developed and commercialized, it will add to the growing roster of SARS-CoV-2 tests that include authorized CRISPR-based tests and current tests that can be performed at the point of care. The group is aiming to develop a molecular test with a one-hour turnaround that exceeds the performance of current at-home antigen tests and that could be ready for a submission for regulatory authorization for SARS-CoV-testing in about two years, said Daniel Brogan, a researcher at UCSD who is one of the CRISPR test developers. NEW YORK ─ University of California San Diego researchers are developing a rapid, CRISPR-based diagnostic test for infectious diseases such as SARS-CoV-2, with the goal of putting more molecular tests into people's hands.
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