Skip to main content
Premium Trial:

Request an Annual Quote

Broad Institute, Collaborators Eye CRISPR Panel to Detect SARS-CoV-2 Variants, Respiratory Viruses


NEW YORK ─ Researchers at the Broad Institute and other organizations have developed a high-throughput, multiplexed CRISPR panel to detect SARS-CoV-2 and emerging SARS-CoV-2 variants, along with other respiratory viruses, with a goal to address some of the limitations of nucleic acid tests and next-generation sequencing platforms.

The collaborators recently published findings from their clinical evaluations in Nature Medicine describing the development and application of the panel to detect SARS-CoV-2, other coronaviruses, and influenza A and B, as well as to identify and differentiate six SARS-CoV-2 variant lineages — Alpha, Beta, Gamma, Delta, Epsilon, and Omicron.

According to Broad Institute test developer and Nature Medicine paper first author Nicole Welch, the group's overall goal is to help close the "massive gap between clinical diagnostics and sequencing."

The new platform, named mCARMEN for microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids, combines CRISPR technology with microfluidics technology. "To our knowledge, mCARMEN is the only diagnostic that combines surveillance capabilities into a single technology platform with the ability to test hundreds of samples in a day for multiple respiratory viruses and variants, while also being able to quantify viral genomic copies," the researchers wrote.

The platform provides a turnaround time of four to five hours, rivaling that of RT-PCR-based SARS-CoV-2 diagnostic tests and far faster, and cheaper, than with sequencing technology used for variants surveillance, Welch added. She noted that mCARMEN detects and identifies variants in less than a day at $13 per sample, around one-tenth the cost of sequencing-based variant identification, which can take seven to 14 days to provide results, too slow for SARS-CoV-2 diagnostic testing and not ideal for variant surveillance.

Overall, the platform's diagnostic panel detects 21 respiratory viruses, allowing the potential for comprehensive testing of patients who present with respiratory symptoms that are difficult to interpret and differentiate, said Welch, a Ph.D. candidate in virology at Harvard University and Pardis Sabeti’s lab at the Broad Institute.

According to the Nature Medicine study, mCARMEN's respiratory panel has demonstrated diagnostic-grade performance in 525 patient specimens in an academic setting and 166 specimens at Massachusetts General Hospital.

It also identified six SARS-CoV-2 variant lineages in 2,088 patient specimens, and the addition of Cas13 and Cas12 enzymes to the panel enabled quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples.

Welch said that the use of integrated fluidic circuits on the commercially available Fluidigm Biomark X microfluidic system and deployment of machine learning to design highly active CRISPR RNA molecules are key to the approach she and her collaborators have developed.

For CRISPR detection, CAS nucleases bind to and use the highly active CRISPR RNA molecules as a guide to target a specific sequence of interest, she said. When a CAS nuclease binds to the target sequence, it cleaves nearby fluorescent reporters which release a light signal indicating the presence of the viral sequence.

In the first proof-of-concept prototype, named CARMEN, developed in 2018, the research group leveraged the Cas13 nucleases to test more than 4,500 crRNA-target pairs on a single array. Cas13-crRNA complexes were separated and confined for barcoding and emulsification prior to pairwise droplet combination for detection by fluorescence microscopy. That approach simultaneously detected 169 viruses in eight samples but had low throughput and required custom imaging chips and readout hardware.

Cameron Myhrvold, a senior author of the Nature Medicine paper, who runs a lab at Princeton University, helped develop the proof-of-concept platform at the Broad Institute.

"When COVID hit we decided to revamp the panel by adding that key virus and looking to develop a version of the technology that could be widely used," Myhrvold said.

As it stood, the platform could be built and used in other labs, but the process "would just require a lot of time and effort," he said.

Paul Blainey, also a senior author of the Nature Medicine study, who runs a lab at the Broad Institute, recommended that the researchers adapt the technology to operate with the Fluidigm microfluidic platform, which automates certain steps. According to Myhrvold, the addition of the Fluidigm platform enabled the automation of otherwise manual processes, such as making and mixing individual emulsions, to prepare them for imaging by microscope.

The Nature Medicine studies demonstrate "the power of combining the mCARMEN platform, utilizing the latest in CRISPR technology, and Fluidigm microfluidics," said Andrew Quong, CSO of South San Francisco, California-based Fluidigm, adding the Biomark X real-time PCR platform "can be easily deployed across the globe for infectious disease surveillance."

mCARMEN now consists of automated RNA extraction, thermocycling for amplification, the microfluidic chips, and a large, benchtop reader — a platform that is similar in size to a large laboratory-based RT-PCR testing system.

With Biomark X, mCARMEN can test a unique combination of either 96 samples using 96 different assays or 192 samples for up to 24 four different viruses or variants. The microfluidic instrument also enabled optimization of the respiratory panel so that it became easy to use and reduced the time to results to less than five hours compared to eight to 10 hours for the original platform, the researchers said.

In the Nature Medicine study, the group compared the performance of mCARMEN to that of its proof-of-concept prototype for detecting synthetic DNA fragments representing the 21 viral targets. Both versions had 100 percent analytical specificity but mCARMEN was 100 percent sensitive to 102 copies/μL and 98.4 percent sensitive to 101 copies/μL while the earlier version was 86 percent and 77.8 percent sensitive, respectively.

Further, in the testing of archived clinical and contrived specimens at Mass General, the respiratory virus panel had 100 percent negative predictive value for all viral targets and greater than 95 percent positive predictive value, except for human metapneumovirus, compared with results from molecular diagnostic assays.

While other clinical investigators validated the platform at Mass General, Welch began to adapt it for the detection of SARS-CoV-2 variants and developed a panel that was integrated with the respiratory panel and didn't "require a massive redesign for detection when a new variant emerged," she said.

The variant panel captures unique mutation patterns, or fingerprints, by detecting a broad array of single nucleotide polymorphisms, she noted, adding that as a result, mCARMEN can quickly flag samples based on a specific fingerprint's pervasiveness, enabling sequencing to determine the variant lineage.

Soon after the Omicron variant emerged, Harvard's CLIA-certified testing laboratory and the Massachusetts Department of Public Health began sending samples to Welch who used mCARMEN to detect the variant.

"A lot of collaborators and third-party connections are interested in using mCARMEN for either respiratory virus detection, emerging variant identification, or in the future to detect blood-borne pathogens," Welch said, noting that the Rhode Island Department of Health, US Centers for Disease Control, US Department of Defense's Defense Advanced Research Projects Agency, and African Centre of Excellence for Genomics and Infectious Diseases are among the collaborators working with Welch and her colleagues.

In the effort to tame the COVID-19 pandemic, a handful of other developers have turned to CRISPR technology to detect the SARS-CoV-2 virus, including the University of California, San Diego, as well as Cambridge, Massachusetts-based Sherlock Biosciences, which 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.

In 2020, the Broad Institute researchers also applied to the FDA for EUA of the respiratory panel as a laboratory-developed test, but the agency said then it had deprioritized tests being conducted in a single laboratory and did not review the application, Myhrvold said.

The group continues to monitor the FDA's priorities and may decide to apply for a new authorization that includes the variant panel, he said, but in the meantime, "we think that there is utility to using the platform in a public health context, which doesn't require the same level of regulatory approval" as an IVD test.