CRISPR-based Assay Platform Detects Multiple Viruses and COVID-19 Variants

A cost-effective virus and variant detection platform has been developed that can test for up to 21 viruses, including SARS-CoV-2, other coronaviruses, and both influenza strains.

Investigators at Broad Institute of MIT and Harvard University (Cambridge, MA, USA) and Princeton University (Princeton, NJ, USA) have described the development of the diagnositic platform known as mCARMEN (microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids). The mCARMEN platform is an upgraded and refined version of their CARMEN system, which depended on nanoliter droplets containing CRISPR/Cas 13-based nucleic acid detection reagents.

Recent computational efforts to identify new CRISPR systems uncovered a novel type of RNA targeting enzyme, Cas13. The diverse Cas13 family contains at least four known subtypes, including Cas13a (formerly C2c2), Cas13b, Cas13c, and Cas13d. Cas13a was shown to bind and cleave RNA, protecting bacteria from RNA phages and serving as a powerful platform for RNA manipulation. It was suggested that Cas13a could function as part of a versatile, RNA-guided RNA-targeting CRISPR/Cas system holding great potential for precise, robust, and scalable RNA-guided RNA-targeting applications.

The original CARMEN platform required custom equipment, involved a manually intensive eight to 10-hour workflow, and offered throughput that was too low for the requirements of a pandemic. Therefore, the investigators modified the CARMEN procedure to work on the Fluidigm (San Francisco, CA, USA) microfluidics and instrumentation platform, making it easier to run and cutting the run time in half. The investigators also streamlined the workflow for greater sensitivity, so that it could detect pathogens in samples with less genetic material. Furthermore, by using CRISPR-based enzymes Cas12 and Cas13 in combination, mCARMEN could not only detect the presence of a virus, but also measured the amount of virus in a sample.

To complement the mCARMEN protocol, the investigators developed a respiratory virus panel (RVP) to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. They further developed an mCARMEN panel to enable identification of six SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens. Finally, they implemented a combined Cas13 and Cas12 approach that enabled quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples.

"The COVID-19 pandemic shows us that we need more testing, more often, particularly early on in a pandemic," said senior author Dr. Cameron Myhrvold, assistant professor of molecular biology at Princeton University. "COVID-19 shows us that challenging viruses will keep emerging, so we have to keep looking for them and come up with better ways of doing that."

The mCARMEN method was described in the February 7, 2022, online edition of the journal Nature Medicine.

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