Researchers at the Broad Institute recognized the need for a single platform with both detection and surveillance capabilities for comprehensive detection of SARS-CoV-2 variants and other respiratory viruses. Currently, there is a trade-off between high-throughput diagnostic methods designed to test large populations, and multiplexed methods that can detect multiple pathogens simultaneously. One of the few clinical diagnostic methods with comprehensive SARS-CoV-2 variant detection is next-generation sequencing (NGS), a method that is time-consuming, expensive, and necessitates bioinformatics expertise to interpret. The Broad Institute’s new platform, microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), was designed to provide cost-effective virus and variant detection.
The mCARMEN platform achieves this through combining CRISPR-based diagnostics and microfluidics with a streamlined workflow. CRISPR-based diagnostics offer an alternative approach to detecting multiple viruses and their variants. By utilizing an optimized workflow on commercially available microfluidic systems, such as Biomark X by Standard Biotools, mCARMEN is highly suitable for clinical use and capable of meeting the public health needs of a viral surveillance platform.
CRISPR, or clustered regularly interspaced short palindromic repeats, are repetitive DNA sequences in bacteria that are transcribed to RNA following viral infection. DNA-cleaving proteins called nucleases are guided by the RNA to cut the viral DNA, which protects the bacteria from the virus. These nucleases are designated as “Cas” for “CRISPR-associated.” The CRISPR-Cas system has many research and clinical applications. In CRISPR-based diagnostics, the effector proteins Cas12 or Cas13 activate upon CRISPR RNA (crRNA) target binding. The target nucleic acids produced by the collateral cleavage activity are then detected via a fluorescent reporter. The crRNA target binding events are highly specific and altered by the presence of sequence variation. This is what allows CRISPR-based diagnostics to differentiate between viruses and viral variants.
Schematic of streamlined mCARMEN workflow
mCARMEN uses Biomark X, a microfluidics technology, which confines fluid flow to mimic a cell’s natural microenvironment. mCARMEN is able to detect up to 96 pathogens simultaneously, detects 169 human-associated pathogens, and can be customized to detect all pathogens with published genomic information.
Three mCARMEN panels, the Respiratory Virus Panel (RVP), Variant Identification Panel (VIP), and the Bloodborne Pathogen Panel (BPP), were developed to test for up to 21 viruses, all SARS-CoV-2 variant lineages, and 20 of the most relevant West African pathogens (16 viruses, 3 bacteria, and 1 parasite, respectively). The first two panels were validated on a combined 2,881 patient samples, and this demonstrated diagnostic capabilities that were on par with or exceeding, currently utilized methods. Increased identification of positive samples, when compared to alternative methods further, suggests that mCARMEN may more robustly protect against sample degradation. The BPP is undergoing further development prior to validation.
Viral quantification using the different reaction times of Cas12 and Cas13
In addition to its capacity to detect multiple pathogens, mCARMEN has the potential to produce clinically relevant data that can inform the care of individual patients. Through utilizing the differing rates of reaction of the Cas12 and Cas13 proteins and the three fluorescent channels detected by the Biomark X, mCARMEN quantifies the pathogen’s genome copies present in a sample. Quantifying the total number of pathogens in a patient’s sample is important for assessing the stage of infection, transmission risk, and most effective treatment plan. Despite this, most of the widely used multiplexed diagnostic approaches do not quantify the pathogens. By incorporating DNA-targeting CRISPR–Cas12 into the Cas13 reaction and using protein-specific reporters in different fluorescent channels, the multiplexing capabilities of mCARMEN were maximized.
mCARMEN is the only detection method 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. As a cost-effective, high-throughput, and multiplexed diagnostic method, mCARMEN will enable the rapid detection of SARS-CoV-2 variants, and holds particular promise in revolutionizing the disease surveillance capabilities of resource-limited countries.
To test mCARMEN in resource-limited countries, the Broad Institute, with the support of WARN-ID, has begun deployment in West Africa. The mCARMEN platform has already been installed at ACEGID in Nigeria and four staff members have been trained on respiratory and bloodborne pathogen panels to detect the most prevalent circulating pathogens in West Africa. Obtaining the necessary equipment for the mCARMEN platform is currently underway at Kenema Government Hospital in Sierra Leone with training and tech transfer planned for the staff later this year.