Since the start of the Covid-19 outbreak, the gold standard for testing has been polymerase chain reaction (PCR). With the first PCR tests emerging as early as mid-January, they have formed an integral part of many countries’ test and trace protocols.
PCR testing – which directly detects the presence of an antigen – has a sensitivity and specificity of more than 95%. It works by amplifying a specific genetic sequence in the virus, making it easier to spot. However, the process is labour-intensive and relatively slow. It requires specialist laboratory equipment, meaning it can’t be deployed the point of care. Nor can it tell you how much virus is present.
There is a clear need for other forms of testing, which can yield results quickly in non-clinical settings. One possible contender is CRISPR, the gene-editing technology. As well as being suitable for use outside the lab, CRISPR-based diagnostics could return results in just five minutes.
“When it comes to clinical diagnostics, where you want to pick up the last bit of RNA that you can find in a nasal sample, I think PCR will potentially stay the standard,” says Dr Melanie Ott, director of the Gladstone Institute of Virology and Professor of Medicine at the University of California, San Francisco (UCSF). “But when it comes to rapid, inexpensive, deployable testing – and also when you want to quantify the virus – I think CRISPR could replace the PCR. It’s a very good supplement.”
How CRISPR testing works
Together with colleagues at UC Berkeley – the bioengineer Daniel Fletcher and the Nobel Prize-winning biochemist Jennifer Doudna – Ott recently reported the proof of concept of a rapid CRISPR assay for Covid-19. Their test was able to correctly identify all Covid-19-positive patient samples within five minutes.
Like other CRISPR tests, it works by programming a CAS enzyme (in this case Cas13a) to act as a ‘scissors’ tool for RNA. It uses a ‘guide’ RNA that complements a specific part of the viral RNA sequence. When the guide RNA binds to the virus, the Cas13a enzyme is activated, cutting apart any nearby RNA and releasing a fluorescent particle that lights up when hit with laser lighting.
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“The whole concept of using CAS as a diagnostic was discovered by Jennifer [Doudna] in 2016,” says Ott. “She found that Cas13 not only cleaves when it’s activated the target RNA, but it also indiscriminately cleaves any RNA that comes close to it. And so when you supply a reporter RNA, that gets cleaved and unquenched and fluorescence evolves.”
While this isn’t the first CRISPR test for Covid-19 – that honour goes to the Sherlock Biosciences test, approved in May – it is the fastest and simplest. Rather than requiring expensive laboratory equipment, it can detect the virus using a mobile phone-based device, built by Daniel Fletcher’s lab.
“We learned that the mobile phone technology has unique opportunities to increase sensitivity,” says Ott. “Initially, we thought, OK, the mobile phone has to become a mini plate reader from the lab – that’s what we usually use to detect the fluorescence. It turns out that the mobile phone is ten times better in its optics than the plate reader. It’s less noisy, it’s more reliable, and we can call a positive earlier.”
Direct detection vs amplification
The Sherlock Biosciences test, made in collaboration with Dr Feng Zhang’s team at MIT, takes around an hour to return results. Like a PCR test, it requires researchers to amplify any viral DNA before running it through the diagnostic, meaning the quantity of virus remains unknown. Without this step, the test simply wouldn’t be sensitive enough. It would require large amounts of virus to yield a positive.
Ott’s test, however, does something radical – it skips the amplification step, and is therefore able to quantify the viral load. This also means reducing cost and complexity, and eliminating that added potential for errors.
Rather than being an afterthought, this ability was baked into the diagnostic from the start. The researchers actually began the device development process two years ago, before Covid-19. They had been hoping to create something different: a point-of-care test for people living with HIV.
“We are entering a phase where HIV-positive people are stopping their immunotherapies to see whether their immune system is strong enough to control the virus,” says Ott. “But for that we need frequent testing of nucleic acids, and CRISPR is a wonderful tool for that. We wanted to combine Jennifer’s CRISPR technology, with Dan’s experience using mobile phone technology as diagnostics, and my interest in virology. We were interested in using the assay to its fullest potential, which means direct detection as opposed to amplification.”
While the team pivoted to Covid-19 in January, they didn’t change their intention. Immediately the question arose – how could they increase the sensitivity of the test so it didn’t require viral amplification? How far could they push the limit of detection?
“What we showed is that you can push it down by a combination of guides, so you don’t rely just on one guide for the CRISPR enzyme, but we can do multiples. And that enhances sensitivity to a point where it becomes interesting,” says Ott.
She is referring to the guide RNA that attaches to the virus. With a single guide, you can to detect 100,000 viruses per microlitre of solution (not ideal for a diagnostic). Introduce a second guide, however, and that falls to 100 viruses per microlitre. This isn’t as sensitive as a PCR test, which only requires one virus per microlitre, but may still be useful for checking the spread of coronavirus in an everyday setting.
“Part of breaking the pandemic is going to be isolating, identifying and isolating people who are infectious very early, so that they cannot spread,” says Ott. “It’s in that rapid testing role that we see our biggest advantage. That’s also where I think knowing whether your viral load is going up or down could be very important, because you’ll know whether you’re going into an infection, or whether you’re at the tail end of an infection.”
The next steps
Currently, the researchers are working to make the test more sensitive, bringing it closer to the PCR gold standard. They are also looking to simplify the system, meaning it can be used at the point of care.
“What you see in the paper is our version two of the assay, which still relies on lab work and can’t be done easily in a drugstore or a doctor’s office,” says Ott. “We have been working on our version three, which is an integrated system where you take the nasal swab, put it into the device, and everything is automated. You can then read the result on your cell phone.”
This is the version of the assay that might be deployed to small businesses, GP surgeries, pharmacies, airports etc. People could be tested on a regular basis, receiving their results within ten to 30 minutes.
A possible version four would remove the phone and integrate its camera into a standalone box. The device would connect with other phones via Bluetooth, improving its cost-effectiveness. However, this may not be the most important factor on the road towards commercialisation.
“One of the things that’s important is speed and time – we’re making a concerted effort to get something out,” says Ott. “We want to bring it into the Berkeley dorms so that the students can test every day and be safe. We want to bring it to Gladstone so that every employee will come to the building and test and be safe. Everybody has their own motivator to make that happen and bring the device out into the community.”
While this won’t be the right test for every setting, it has scope to be a useful alternative to PCR. Ott and her fellow researchers are excited about the prospects, and hope to make a difference in the near future.