Now, in a new study, researchers from Harvard University's Wyss Institute for Biologically Inspired Engineering, the Massachusetts Institute of Technology, and several Boston-area hospitals have developed an inexpensive, CRISPR-based diagnostic test that allows users to test themselves for SARS-CoV-2 and its many viral variants at home, using saliva samples, without the need for additional instrumentation. The results of the study are published in the August 6, 2021 issue of ScienceAdvances in a paper titled "MinimallyinstrumentedSHERLOCKforCRISPR-basedpoint-of-careiagnosisofSARS-CoV-2andembolism. CoV-2andemergingvariants."
The diagnostic device, called MinimallyInstrumentedSHERLOCK, is easy to use and provides results that can be read and verified by a companion smartphone app within an hour. It successfully distinguished three different variants of SARS-CoV-2 in experiments and can be quickly reconfigured to detect additional variants, such as the Delta variant. The device can be assembled with a 3-D printer and commonly available parts for about $15, and reusing the parts brings the cost of a single detection down to $6.
Helenade Puig, the paper's *** co-first author and a postdoctoral researcher at Harvard University's Wyss Institute for Biologically Inspired Engineering and the Massachusetts Institute of Technology, said, "miSHERLOCK eliminates the need to transport patient samples to centralized testing sites and greatly simplifies the sample preparation step, allowing patients and physicians to more quickly and accurately understand the health status of individuals and communities, which is critical in an evolving pandemic."
From Supply Chain to SHERLOCK
As a pediatrics instructor specializing in infectious diseases at Boston Children's Hospital, the paper***s co-first author, Dr. RoseLee, has been on the front lines of the COVID-19 pandemic for more than a year. Her experience at the clinic provided the inspiration for the eventual development of the miSHERLOCK program.
Lee says, "Simple things that used to be readily available in hospitals, such as nasopharyngeal swabs, suddenly became difficult to obtain, so routine sample handling procedures were disrupted, which is a big problem in a pandemic context. Our team's motivation for this project was to remove these bottlenecks and provide an accurate diagnostic for COVID-19 without relying too much on the global supply chain, and also to accurately detect the viral variants that are starting to emerge."
For the SARS-CoV-2 detection portion of their diagnostic device, these authors chose a CRISPR-based technology built in the lab of Dr. Jim Collins, the paper's corresponding author and a core faculty member at Harvard University's Wyss Institute for Biologically Inspired Engineering, known as SHERLOCK. SHERLOCK uses CRISPR's "molecular scissors" to cut DNA or RNA at specific sites, which has an added benefit: This particular type of molecular scissors also cuts other DNA fragments in the surrounding region, allowing it to cut single-stranded nucleic acid probe molecules that carry fluorescent signals, which in turn generates fluorescent signals indicating that the target has been successfully cut.
The diagnostic procedure requires only that the user spit into the sample preparation chamber, then transfer the collection tray to the reaction chamber and press the plunger to activate the reaction and minimize the risk of cross-contamination. Image from ScienceAdvances, 2021, doi: 10.1126/sciadv.abh2944.
These authors created a SHERLOCK assay designed to cleave a specific region of the gene encoding the nucleoprotein of SARS-CoV-2 that is conserved in multiple variants of the virus. When this molecular scissor, called Cas12a, successfully binds and cleaves the NP gene, a nearby single-stranded DNA probe is also cleaved, resulting in a fluorescent signal. They also created additional SHERLOCK assays designed to target a set of viral mutations in the spiking protein sequences that represent the three SARS-CoV-2 viral variants: Alpha, Beta, and Gamma.
With assays that could reliably detect the viral RNAs within the range of acceptable concentrations for diagnostic tests licensed by the U.S. Food and Drug Administration methods, these authors next focused on perhaps the most difficult challenge in diagnostics: sample preparation.
Spit, wait, scan
Dr. XiaoTan, the paper's ****ing co-first author and a clinical researcher at Harvard University's Wyss Institute for Biologically Inspired Engineering, says, "When you test for nucleic acids in a sample, you need to do a lot of steps to prepare the sample so that you can actually extract and amplify those nucleic acids. You have to protect the sample as it's being transported to the testing facility and also make sure it's not infectious if you're dealing with a transmittable disease. In order to make it a really easy-to-use diagnostic test, we had to simplify it as much as possible."
These authors chose to use saliva rather than nasopharyngeal swab samples as their collection method because it's easier for the user to collect saliva and because it's been shown that SARS-CoV-2 can be detected in saliva for many more days after infection. But untreated saliva poses its own challenges: it contains enzymes that degrade a variety of molecules, producing a high rate of false positives.
These authors developed a new technique to address this problem. First, they added two chemicals called DTT and EGTA to saliva and heated the samples to 95°C for 3 minutes, which eliminated false-positive signals in untreated saliva and cleaved any virus particles. They then added a porous membrane in order to capture the RNA on its surface, which can finally be added directly to the SHERLOCK reaction to produce the assay.
To integrate saliva sample preparation and the SHERLOCK reaction into one diagnostic device, these authors designed a simple battery-powered device with two chambers: a heated sample preparation chamber and an unheated reaction chamber. The user spits into the sample preparation chamber, heats it, and waits three to six minutes for the saliva to enter the filter. The user removes the filter and transfers it to the column of the reaction chamber, then presses the plunger, places the filter into the chamber, and punctures the reservoir to activate the SHERLOCK reaction.After 55 minutes, the user observes the reaction chamber through a tinted transilluminator window to confirm the presence of a fluorescent signal. They can also use a companion smartphone application that analyzes pixels recorded by the smartphone camera to provide a definitive positive or negative diagnosis.
These authors tested their diagnostic device with clinical saliva samples from 27 COVID-19 patients and 21 healthy patients, and found that miSHERLOCK correctly identified COVID-19-positive patients 96 percent of the time, and patients without the disease 95 percent of the time. They also tested the diagnostic device's performance in detecting SARS-CoV-2Alpha, Beta, and GammaSARS-CoV-2 variants by adding full-length synthetic viral RNA containing mutations representative of each viral variant to the saliva of a healthy person and found that the device was effective at various viral RNA concentrations.
Devora Najjar, co-lead author of the paper*** and a research associate in the Collins lab, said, "One of the strengths of miSHERLOCK is that it is completely modular. The device itself is separate from the assay, so you can round into different assays to detect the specific RNA or DNA sequence you want to detect. The device costs about $15, but mass production will bring that down to about $3. Assays for new targets can be established in about two weeks, allowing rapid development of new variant assays for COVID-19 and other diseases."
Ready for real-world applications
These authors built their diagnostic device with low-resource environments in mind, as the pandemic has brought to light the vast inequalities in healthcare between different parts of the world. The components of the device can be built by anyone with access to a 3-D printer, and the associated information files and circuit designs are publicly available online. The addition of a smartphone application is also aimed at resource-limited environments, as cell phone service is available almost anywhere in the world, even in areas that are difficult to reach on foot. They are eager to work with manufacturers who are interested in mass-producing miSHERLOCK for global distribution.
Collins said, "When the miSHERLOCK program began, little SARS-CoV-2 variant monitoring was done. We knew that this viral variant tracking would be important in assessing the long-term impact of COVID-19 on local and global communities, so we pushed ourselves to build a truly decentralized, flexible, and user-friendly diagnostic platform. By addressing sample preparation, we've ensured that this device is virtually ready for consumer use, and we're excited to work with industrial partners to commercialize it."
Dr. DonIngber, founding director of the Wise Institute for Biologically Inspired Engineering at Harvard University, said, "By combining cutting-edge biotechnology with low-cost materials, this team of researchers has constructed a powerful diagnostic device that can be made and used locally by people without advanced medical degrees. It's a perfect example of the mission of Harvard's Wyss Institute for Biologically Inspired Engineering: to put life-changing innovations into the hands of people who need them."
References:
HelenadePuigetal.MinimallyinstrumentedSHERLOCKforCRISPR-basedpoint-of careiagnosisofSARS-CoV-2andemergingvariants.ScienceAdvances, 2021, doi: 10.1126/sciadv.abh2944.