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UCSD Developing SARS-CoV-2 At-Home, Molecular Test Leveraging CRISPR, Isothermal Amplification

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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.

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.

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.

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.

According 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. 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.

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.

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.

As a result, he and his colleagues are using a CRISPR enzyme subtype, Cas13d, to compensate for a lack of specificity.

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.

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.

"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."

Like other CRISPR-Cas13 enzymes, Cas13d initially cleaves target RNA and subsequently cleaves bystander RNA in the vicinity. 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. When a pathogen is not in the sample, no cleavage occurs, signaling a negative test result.

Cas13d has a preference for a sequence designed into the modified reporter probe, and each reporter can be designed to detect a specific genomic sequence. "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.

According 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.

Still, there are important near and intermediate term obstacles that could hinder the deployment of such tests, including access to reagents "that, due to proprietary and commercial factors, still create bottlenecks," said Castro, who is not affiliated with UCSD but is leading the development of CRISPR-based detection of the human papillomavirus for cervical cancer screening.

Regardless, several firms are developing, or have developed, CRISPR-based diagnostic tests to detect SARS-CoV-2.

For example, Sherlock Biosciences has developed and commercialized its SHERLOCK platform, named for Specific High-sensitivity Enzymatic Reporter unlocking, which combines enzymes such as Cas13, Cas12a, and Csm6 to allow for the simultaneous detection of multiple nucleic acids. The firm announced in May 2020 that it had received Emergency Use Authorization from the US Food and Drug Administration for its Sherlock CRISPR SARS-CoV-2 kit designed for use in high-volume CLIA laboratories and hospitals.

Additionally, Sherlock and Mammoth Biosciences have signed partnership deals with other firms — Sherlock Bio with Binx Health and Mammoth with the consumer healthcare arm of pharmaceutical company GlaxoSmithKline — to develop and commercialize rapid CRISPR-based point-of-care or home-use diagnostic tests for COVID-19.

Because UCSD's technology could potentially address other medical indications, the group is exploring its use to detect the dengue virus, Brogan said. However, UCSD doesn't want to be limited to "just one or two diseases," he said, adding, "We think this technology is more of a platform that is generalizable for many conditions."

The group has some challenging work ahead of it to move the platform closer to commercialization. The system has been developed to use nasopharyngeal samples for testing, but the researchers are interested in developing it to test saliva samples, Brogan said.

The UCSD researchers aim to provide a SARS-CoV-2 test that costs less than $5, which should make it easily adoptable for home use, Brogan said, adding, "Though we are not there yet, we've taken a big step toward putting CRISPR diagnostic tests into people's hands."

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