More than three months into the coronavirus pandemic, widely acknowledged shortcomings with testing continue to hamper the nation’s recovery. So when a team of microbiologists headed by Dr. Feng Zhang of the McGovern Institute at MIT and the Broad Institute reported this month that they had ironed out a protocol for a simple, cheap, point-of-care test that uses CRISPR to detect the virus, it was hailed as one of the most important contributions to fighting COVID-19.
Efforts to build and scale up a diagnostic have been beset by a number of snags, from a scarcity of chemicals called reagents and equipment to slow return of results. As officials debate how to safely reopen the country, those weaknesses would need to be rectified in a way where relaxing stay-at-home orders doesn’t set off a viral rebound.
According to the Harvard Global Health Institute, in order to safely reopen the country and keep it open, we must ramp our testing rate from about a million per week to a million per day. But that remains a stretch by conventional means.
Enter CRISPR, the precision genome-editing technique that is anything but conventional.
Dr. Zhang and his colleagues harnessed a new type of CRISPR to build a test able to rapidly detect as few as 100 coronavirus particles in a swab or saliva sample, according to instructions for the new test, called STOPCovid, that they posted online. The team’s focus has now shifted to proving that the test is safe and effective on a mass scale.
“Like any diagnostic chemistry, we need to demonstrate that STOPCovid is accurate on a large enough cohort of patient samples to provide benefit,” wrote Drs. Jonathan Gootenberg and Omar Abudayyeh, both of the McGovern Institute, in response to emailed questions. “In addition, the current pandemic has made clear the need to scale to thousands or millions of tests, and solutions for that are necessary as well.”
As to when the new diagnostic may be pressed into service against the novel coronavirus, “It’s hard to predict what the timeframe would be for a point-of-care or at-home test,” the two noted. “But given the need for these diagnostics, we would hope at latest by the end of the year.”
That timeframe is possible because a prototype for a quick, easy, cheap and precise CRISPR-based diagnostic test had already existed. In 2017 Dr. Zhang and bioengineer Dr. James Collins published research showing that CRISPR could be trained to detect extremely low amounts of genetic material and was suitable for use during disease outbreaks. They dubbed this system Sherlock, for specific high-sensitivity enzymatic reporter unlocking.
It’s from that earlier prototype that the new SARS-CoV-2 test takes its cue. STOP stands for Sherlock Testing in One Pot – think multiple steps reduced to a single reaction in a tube. It’s similar to DNA-targeting CRISPRs, like the well-known Cas9 system for DNA-editing of the human genome. Only this system has been reformulated to target strands of RNA, which are the building blocks of viruses.
Once a guide RNA molecule brings the Cas12 enzymes to the area of interest, the enzymes cut it in such a way that it generates a fluorescent readout. That readout is detectable, in much the same way home pregnancy tests pick up on pregnancy-related hormones.
Broadscale testing of the type afforded by STOPCovid would be integral to reopening the economy again, said Dr. Neville Sanjana, a genome engineer who was not involved in the CRISPR diagnostic research but whose lab, based at the New York Genome Center and NYU, is involved in several efforts using CRISPR, both COVID-related and non.
“The CRISPR-based diagnostics do present a path forward not just to scaling up but to longitudinal sampling, continual sampling,” said Dr. Sanjana, who, in addition to being a faculty member at the New York Genome Center, is also assistant professor of biology at New York University and of neuroscience and physiology at the NYU School of Medicine. “We’re only going to feel comfortable going back to work if not only do you know that your coworker was Covid-negative yesterday, but you know your coworker was Covid-negative last week and Covid-negative today – if you have as many assurances as possible.”
To fully appreciate why a CRISPR-based test might be a good fit in this situation requires a comparison to the standard COVID-19 test, which is based on a technology called qPCR. The qPCR tests work by detecting nucleic acids, like RNA, and amplifying them. In that sense, they’re similar to CRISPR-based tests.
However, that’s where the similarities end.
The qPCR tests require a specialized piece of equipment combining a thermocycler (a machine that automatically cycles through multiple temperature changes) and a microscope. These machines are typically only found in labs and normally cost a few thousand dollars apiece. The other big limitation of qPCR is that the reagents needed to perform the test require cold storage.
CRISPR-based diagnostics, on the other hand, can assay the presence or absence of a nucleic acid at just one temperature. In addition, it’s been demonstrated – also by Dr. Zhang and colleagues – that it’s possible to freeze-dry CRISPR enzymes, obviating the need for cumbersome cold storage.
“You don’t need the microbiology lab. Instead, you maybe need a stove,” said Sanjana.
“One of the unique aspects of CRISPR is that it enables sensitive detection without requiring fancy equipment or refrigerated storage.”
Reading the results is relatively simple, too. Whereas qPCR and PCR-based test results must be read out either using a microscope or something called agarose gel electrophoresis – another lab mainstay – the CRISPR-based assay for SARS-CoV-2 uses a lateral flow strip for readout, akin to a home pregnancy test.
A test strip is placed in a tube, and the presence of two lines indicates SARS-CoV-2. Results come in about an hour, the researchers say, with no special handling needed. A mobile phone app can analyze images captured by the phone camera to readout test results, they noted in their white paper.
“That [kind of straightforward readout] would be an enabling technology,” said Sanjana, even in places located close to a traditional lab.
What’s more, STOPCovid tested on a nasopharyngeal swab bested sensitivity and specificity rates of qPCR, according to Zhang’s team (it’s been shown to work in saliva, too). His group has prepared reagents for 10,000 tests to make freely available to other researchers who want to evaluate its diagnostic use.
At least two other research groups are studying CRISPR technology’s promise in diagnosing coronavirus. The FDA was suitably impressed with the test coming out of the Broad group, which is being commercialized through a company aptly named Sherlock, that the agency handed it an “emergency use authorization” this month. Meanwhile, Zhang is reportedly talking to would-be commercial partners about designing a device based on a disposable, single-use test cartridge, similar to a pregnancy test.
“That’s the kind of thing we want,” said Dr. Sanjana. “If I had to think about what would be the ideal test, it would be something that would be dirt cheap, you could do it every single day, you could test yourself and your whole family, and you’d get results instantly or close to instantly. This is what we need to go back to work safely.
“Not to say that the CRISPR-based testing is the end-all-be-all,” he added, “but I think it can fill a very unique niche, even in places where you might have access to what you need to do qPCR-based tests. But the convenience would be greater and thus the adoption would be better.”