Skip to main content
Premium Trial:

Request an Annual Quote

CRISPR-Based Platform Tests for SARS-CoV-2 Using Saliva Samples

Premium

NEW YORK – Researchers at the Wyss Institute for Biologically Inspired Engineering, the Broad Institute, MIT, and elsewhere have developed a CRISPR-based, low-cost, point-of-care diagnostic that they said can detect SARS-CoV-2 from unprocessed patient saliva in about one hour.

The new assay, called miSHERLOCK, is based on the SHERLOCK, short for specific high-sensitivity enzymatic reporter unlocking, and DETECTR, for DNA endonuclease-targeted CRISPR trans reporter, gene editing platforms. These platforms, which use the Cas13a or Cas12a nucleases, have been used to create several ultrasensitive molecular diagnostics for a variety of targets, including infectious diseases such as Zika virus. Over the course of the COVID-19 pandemic, they've also been shown to be useful in creating CRISPR-based diagnostics for SARS-CoV-2 in a variety of settings.

In their paper, published on Friday in Science Advances, the researchers showed that miSHERLOCK takes unprocessed patient saliva, then extracts, purifies, and concentrates the viral RNA, performs amplification and detection reactions, and provides a fluorescent visual results output with only three user actions and in one hour. They were able to achieve highly sensitive multiplexed detection of SARS-CoV-2 and mutations associated with the Alpha, Beta, and Gamma variants.

They also noted that the platform's modular design makes it easy for a user to change assays depending on need and can be rapidly reconfigured to detect different viruses and variants of concern. Further, a smartphone app enables output quantification, automated interpretation, and the possibility of remote, distributed reporting of results.

According to first author and MIT researcher Helena de Puig, the technology's adaptability makes it possible to rapidly build assays for new variants of concern as they crop up, as well.

"Variant detection is performed via sequencing, which provides the complete genomic information from any specific sample. Only a very small subset of COVID-positive samples are actually sequenced due to the expense and effort required, even in resource-rich settings," she wrote in an email. "Due to the number of infections in the ongoing pandemic, this is still sufficient to identify mutations in variants of concern, which link viral genotypes to particular outbreaks or clinical situations."

She and her colleagues use those key mutations to develop specific point-of-care assays, taking around one to two weeks to develop high-performance assays for any variant.

What makes miSHERLOCK so useful for the point of care, the researchers noted, is that the platform integrates an optimized one-pot SHERLOCK reaction with an RNA paper-capture method compatible with in situ nucleic acid amplification and Cas detection. It combines instrument-free, built-in sample preparation from saliva, room temperature stable reagents, battery-powered incubation, and simple visual and mobile phone-enabled output interpretation with a limit of detection that matches US Centers for Disease Control and Prevention RT-qPCR assays for SARS-CoV-2 of 1,000 copies/ml.

And although saliva is not a commonly used clinical sample, several studies have demonstrated comparable performance between saliva and nasopharyngeal samples for the detection of SARS-CoV-2, they added. Further, in paired collection samples in hospitalized patients, salivary SARS-CoV-2 viral load has been shown to be marginally higher than nasopharyngeal swabs and positive for a greater number of days.

De Puig said the researchers currently envision two use-scenarios for miSHERLOCK. In resource-rich settings, the platform could be deployed as an easy-to-use, low-cost, fast, and accurate SARS-CoV-2 test.

"Specific mutations and variants have been associated with altered efficacy of particular treatments and viral transmission, even after vaccination with current vaccines," she said. "Although current vaccines still offer good protection against hospitalization and death from COVID-19, it is certainly possible that future variants will not be as well-covered. Knowing whether a person has SARS-CoV-2 infection and what strain is involved could have major effects on a patient's recommended course of action and treatment. Using the companion smartphone app, this could be set up to automatically report results to a school or employer if desired since many now require scheduled COVID testing."

In low-resource settings, she added, they envision miSHERLOCK being deployed by health departments as a method to decentralize strain testing and tracking in remote areas. For example, when they were developing the platform, a key variant of concern was the Gamma variant, which was originally identified in Brazil. An epicenter of infections was Manaus, in the middle of the Amazon rainforest.

"Through the use of our entirely self-contained device and the companion smartphone app, we can take advantage of the wide availability of mobile service even in remote areas to provide real-time SARS-CoV-2 strain detection and reporting back to the health ministries," de Puig said. "This circumvents the need to rely on less than robust transportation systems and the additional handling of potentially infectious samples. The information on the distribution of specific strains may also guide further public health efforts and the allocation of resources."

In the miSHERLOCK diagnostic workflow, the researchers wrote, the user introduces 2 ml of saliva into the collector, which contains preloaded lysis reagents. The user activates the heater on the device, and waits three to six minutes, which allows enough time for the viral particles to be lysed, the salivary nucleases to be inactivated, and the saliva to be wicked into the filter, leaving concentrated purified RNA behind on a membrane. The user then removes the collector and transfers the sample preparation column to the reaction chamber, pushes a plunger into the column, which punctures a water reservoir to rehydrate and activate the SHERLOCK reaction, and deposits the membrane inside the reaction chamber. Then the user can come back in 55 minutes to observe the visual fluorescence readout.

The companion phone app provides automated quantitation and simplified interpretation of SHERLOCK results and uses the embedded camera in a smartphone in combination with a color segmentation algorithm to detect and quantify the observed fluorescence at the end of the incubation period.

De Puig believes that miSHERLOCK could present patients with the best option for at-home testing of SARS-CoV-2, and eventually, many other pathogens as well. While there are other home-based test kits, many are antigen-based tests, "which are generally far less sensitive than nucleic acid tests," she said. Further, most of the tests capable of identifying SARS-CoV-2 genes are self-collection kits that are mailed back to a central testing facility and typically require 48 to 72 hours, and can cost upward of $120, she added.

The full miSHERLOCK device, including all testing components, costs $15, de Puig said. That could come down to $6 with reuse of the housing and electronics, and could be as low as $2 to $3 per test if produced at scale.

The researchers currently have no plans for commercialization, but de Puig said they'd welcome a collaboration with a commercial partner, if one presented itself.

In addition to being able to create assays for new variants of concern, the researchers are working on using miSHERLOCK for the detection of other diseases, as well.

"Changing the target is as simple as changing two primers and the guide RNA, and any nucleic acid could be targeted," de Puig said. "Other common infectious diseases detectable in saliva include hepatitis C virus and human papillomavirus. The modular nature of miSHERLOCK also allows for its adaptation for other samples such as urine, blood, or other bodily fluids."

They're currently adapting the device to diagnose febrile illnesses, such as malaria or dengue in low-resource settings, as well as pathogens in water supplies.

The Scan

Booster for Kids

The US Food and Drug Administration has authorized a booster dose of Pfizer-BioNTech's SARS-CoV-2 vaccine for 5-to-11-year olds, the Washington Post reports.

Novavax Anticipates Authorization

Novavax expects to receive US Food and Drug Administration authorization for vaccine soon, according to CNBC.

Some Old Microbes Indeed

Vice reports that researchers may have uncovered 830-million-year-old microbes.

PNAS Papers on Meiotic Crossover, RNA Targeting, SNAT7 mTORC1 Regulation

In PNAS this week: meiotic crossover formation in C. elegans, approach to identify small molecules affecting RNA activity, and more.