Researchers Employ Novel CRISPR Tech to Accelerate COVID-19 Testing

By Pavel Ryzhov, Ph.D.

There is an acute shortage of data about the community spread of COVID-19 pandemic. Hence, among the many coronavirus diagnostic tests that are currently deployed in the US and across the world, the rapid Point-of-Care (POC) tests are getting the most attention. However, due to the difficulties in the supply chain of the reagents, sample handling difficulties, and the cost of operation, the deployment of these tests has been stalled or significantly slowed down.

To address these scalability and accessibility issues, a team of researchers headed by Prof. Feng Zhang and collaborators Jonathan Gootenberg and Omar Abudayyeh at the McGovern Institute for Brain Research at MIT have been working on the development of a new platform for diagnostics called STOP (SHERLOCK Testing in One Pot) 1,2. This platform aims to provide rapid results (under 70 min) after taking saliva or nasopharyngeal swabs from patients.

The protocol harnesses high fidelity nucleic detection technology called SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) that was previously developed by the same team of researchers. To achieve that, the viral RNA is first amplified by isothermal amplification reaction and then detected by CRISPR-mediated reporter unlocking. Unlike other previously developed CRISPR-based tests that rely on dual reaction steps like liquid handling and opening of tubes, one important differentiator of this novel test is its simple testing chemistry. In addition, as compared to more common RT-qPCR tests, STOPCovid also frees the POCs from relying on the expensive equipment and sparse reagents.

Image courtesy: STOPCovid website

Specifically, the authors leveraged loop-mediated isothermal amplification (LAMP) reaction for the RNA amplification, which is also compatible with the downstream CRISPR reaction due to the buffer compatibility between the two. Furthermore, to account for an increase in temperature to perform LAMP as compared to other recombinase polymerase amplification-based tests, the authors did the following. They optimized for thermostability of Cas enzyme by selecting Cas12b from Alicyclobacillus acidiphilus and matched it with a single-guide RNA from a close ortholog of Cas12b from Alicyclobacillus acidoterrestris to provide a CRISPR array for increased nuclease activity as compared to previously published one.

In addition, 94 reaction components were screened for improvements in the thermal stability of the STOP test, with taurine found to be significant for the kinetics of the reaction. The limit of detection (LOD) for the test was found to be 100 copies of SARS-CoV-2 and highly reproducible: 3/3 replicates for 11/12 positive patient samples with at least 2/3 replicates for 12/12 samples and all correctly identified negative patient samples (5 patients, 3 replicates each). Finally, the authors claim that reaction components can be stored and maintained even after 6 cycles of freezing and thawing.

Since the authors have not yet obtained FDA authorization for the clinical application of this test, it is only currently available for research purposes. Nevertheless, it represents an important step in the POC test development. The low cost of the equipment required for the test (swabs, kit, and a source of heat), elimination of extra steps, along with reproducibility and sensitivity, are the key metrics that are crucial for expanding the current testing capacities in the US and elsewhere. Therefore, the authors point out that they are planning to collaborate with other researchers to advance this diagnostics platform.

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References

1. http://news.mit.edu/2020/sherlock-based-one-step-test-provides-rapid-sensitive-covid-19-detection-0505
2. https://www.stopcovid.science/docs/STOPCovid%20Whitepaper.pdf

Author

Pavel Ryzhov