CRISPR-Based COVID-19 Test Identifies Variants from Saliva Without Lab Analysis

CRISPR-Based COVID-19 Test Identifies Variants from Saliva Without Lab Analysis

Point-of-care COVID-19 tests have been developed that can be used to rapidly identify the presence of the SARS-CoV-2 virus. These tests are simple to administer and can be used by individuals to perform tests at home. However, it has become increasingly important to be able to identify the strain of virus involved, due to the increase in new variants that make current vaccines less effective. Rapid identification of new infections with these variants is essential to prevent their spread.

Currently, while there are diagnostic tests that can be conducted to identify different variants from patient samples, to identify specific variants samples must be sent to clinical laboratories to be genetically sequenced, which increases the time between test and diagnosis and significantly adds to the cost.

Now a new method of testing has been developed by researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Massachusetts Institute of Technology (MIT), in collaboration with Boston-area hospitals. The new technique has been incorporated into a new diagnostic test that can be performed by individuals in their own homes from saliva samples, which requires no extra instrumentation other than the test kit and does not need samples to be sent to the lab for analysis.

The diagnostic test can be manufactured using a 3D printer using components that cost around $15, and the individual assay cost is around $6. Since saliva samples are used for the test, they can be easily collected by patients themselves at home. The sample is placed in the test and a result is generated in about an hour which can be read by the patient and confirmed using a smartphone app. The entire test kit is around 2 cm x 5 cm x 5.5 cm.

The CRISPR-based test – named Minimally Instrumented SHERLOCK (miSHERLOCK) – was shown to be capable of not only detecting SARS-CoV-2, but was also able to distinguish between three different SARS-CoV-2 variants. The test could also be easily altered to identify other SARS-CoV-2 variants.

“miSHERLOCK eliminates the need to transport patient samples to a centralized testing location and greatly simplifies the sample preparation steps, giving patients and doctors a faster, more accurate picture of individual and community health, which is critical during an evolving pandemic,” said Helena de Puig, Ph.D., Postdoctoral Fellow, Wyss Institute and MIT and co-first author of the paper.

miSHERLOCK is based on CRISPR technology – Specific High-sensitivity Enzymatic Reporter unlocking (SHERLOCK) – developed in the lab of Wyss Core Faculty member Jim Collins, Ph.D. CRISPR is used to make cuts to DNA or RNA in order to replace or modify sections of genetic code, usually to correct mutations, but can also be used in diagnostic tests.

SHERLOCK takes advantage of the ability of the CRISPR system to make cuts to RNA and DNA at specific locations, but since the system can be used to make cuts to other pieces of DNA in the surrounding area, it has been engineered to include nucleic acid probe molecules that are capable of generating a signal when a targeted section of the genetic material has been cut.

In the test, the Cas12a enzyme binds to a specific section of the SARS-CoV-2 RNA corresponding to the Nucleoprotein gene, which is conserved across multiple variants of the SARS-CoV-2 virus. Single stranded DNA probes are also cut, which generate a detectable fluorescent signal if the Nucleoprotein gene has been cut, which will give either a positive or negative result. Additional assays were developed that focused on the spike protein sequences, which could detect the Alpha, Beta, and Gamma variants of SARS-CoV-2 within the same test. The test has a sensitivity within the range of current tests authorized for use by the U.S. Food and Drug Administration (FDA).

One of the problems with testing for SARS-CoV-2 in the early stages of the pandemic was a global shortage of nasopharyngeal swabs, which created testing bottlenecks. The researchers opted for a method of sampling that would not involve these bottlenecks and would also be easy for patients to administer themselves. The researchers choose to detect the virus from saliva samples, as studies have shown SARS-CoV-2 is detectable in saliva, and for more days post-infection than samples collected using nasopharyngeal swabs.

One problem is saliva contains enzymes that degrade certain molecules, which can result in a high number of false positives. The researchers solved this problem by developing a new technique involving the use of the chemicals DTT and EGTA, which are added to the saliva and are then heated to 95°C for 3 minutes. The researchers found this eliminated the false positives and also sliced open viral particles. A porous membrane was then created to trap RNA on its surface, which could be added directly to the SHERLOCK reaction to generate a result.

The generation of heat for the diagnostic test was solved by using a battery powered device with two chambers, one for the unheated reaction chamber and one that was heated to prepare the sample. Patients simply spit into the prep chamber, turn on the heat, and then wait between 3 and 6 minutes while the saliva is wicked into the filter. The filter is then removed and transferred to the reaction changer and a plunger is pushed to deposit the filter in the chamber. A water reservoir is then punctured to activate the SHERLOCK reaction.

A tinted transilluminator window allows the patient to see into the reaction chamber and can confirm whether there is a fluorescent signal after 55 minutes. A smartphone app has also been developed that can be used to analyze the pixels from an image of the chamber to confirm a positive or negative diagnosis.

miSHERLOCK device: A CRISPR-enabled POC diagnostic that integrates mechanical, electronic, biochemical, and optical components. Source: Helena de Puig et al.

In tests on samples of saliva from 27 COVID-19 patients and a control group of 21 healthy patients, the test was shown to identify 96% of COVID-19 patients, and also found to be effective at identifying the Alpha, Beta, and Gamma SARS-CoV-2 variants at varying RNA concentrations.

“One of the great things about miSHERLOCK is that it’s entirely modular. The device itself is separate from the assays, so you can plug in different assays for the specific sequence of RNA or DNA you’re trying to detect,” said MIT Media Lab research assistant co-first author Devora Najjar. “The device costs about $15, but mass production would bring the housing down to about $3. Assays for new targets can be created in about two weeks, enabling the rapid development of tests for new variants of COVID-19 and other diseases.”

You can read more about the study in the paper – Minimally Instrumented SHERLOCK (miSHERLOCK) for CRISPR-based Point-of-Care Diagnosis of SARS-CoV-2 and Emerging Variants – which was recently published in Science Advances. DOI: 10.1126/sciadv.abh2944.