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COVID Assays Using CRISPR, Smartphones Show Potential for At-Home Testing

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NEW YORK – Two different research groups have developed new CRISPR-based diagnostic assays for the SARS-CoV-2 virus that can be read with smartphones, and can quantitatively measure patients' viral loads in addition to testing for infection.

Though the assays differ somewhat in their technological design and currently accepted sample type, both tests are meant to function as a one-step process, without the need for separate RNA extraction or amplification. The researchers who developed the assays are hoping that these features can eventually make the tests easy for consumers to use at home.

In a paper published in Cell earlier this month, researchers led by University of California, Berkeley professor and CRISPR pioneer Jennifer Doudna, Gladstone Institute of Virology Director Melanie Ott, and UC Berkeley bioengineer Daniel Fletcher described their assay, which uses CRISPR-Cas13a for direct detection of SARS-CoV-2 RNA in nasal swabs. The assay achieved sensitivities of about 100 copies per microliter in less than 30 minutes of measurement time without the need for amplification, and accurately detected pre-extracted RNA from a set of positive clinical samples in less than five minutes.

In a paper published in Science Advances the following week, investigators led by researchers at Tulane University School of Medicine described their own smartphone-read CRISPR assay for quantitative detection of SARS-CoV-2 from saliva samples. The assay uses the Cas12a enzyme to enhance the signal from an amplified viral RNA target, and the researchers reported a 15-minute sample-to-answer time that does not require RNA isolation or laboratory equipment.

Ott and her colleagues chose to use Cas13 because the nuclease directly targets and cleaves RNA, obviating the need for reverse transcription of the RNA into DNA and then amplification by PCR, as is done in current standard tests.

Bo Ning, first author on the Science Advances paper and an assistant professor at Tulane, said he and his colleagues chose CRISPR-Cas12a combined with recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique, as the basis for their assay. RT-RPA, a simpler and less instrument-heavy alternative to RT-PCR, does the job of transcribing the viral RNA to DNA for Cas12a to cleave and detect. The enzyme does the rest, Ning said.

Despite the differences in the functions of their enzymes, however, the two assays do have their simplicity and one-step ethos in common. "All this reaction is in one step," Ning added. "It's all integrated — the reagents, the detection probes, everything in one solution."

A quantitative assay

Ott and her colleagues combined different CRISPR RNAs (crRNAs) targeting SARS-CoV-2 RNA with the Cas13 to improve their assay's sensitivity and specificity, and were able to directly quantify the viral load in their sample using enzyme kinetics. They also added an RNA-based probe that, when cleaved, produces fluorescence as a positive viral signal.

They're currently working on integrating it with a smartphone-based reader, Ott said. She also noted that since writing its paper the team has integrated the RNA extraction step directly into the assay so the test can now directly analyze a sample rather than requiring pre-extracted RNA.

"We started this two years ago when we were trying to develop this assay for HIV, as an at-home assay for people who want to get off anti-retrovirals," Ott said. "So, we decided from the beginning that we would not want to amplify."

The reason the assay yields quantitative RNA measurements rather than just a simple positive or negative result is because it doesn't require pre-amplification of the viral genome. The combination of multiple crRNAs increased the activation of Cas13a, allowing the researchers to analyze the change in the test's fluorescent signal over time, rather than solely its endpoint fluorescence. The use of multiple crRNAs that target different parts of the viral genome can also safeguard against a potential loss of detection due to naturally occurring viral mutations, the researchers noted.

Obviating amplification also makes the assay easier to use and more suitable for home testing, Ott said.

In the future, the investigators anticipate further improving the assay by systematically searching for the best crRNA combinations across the entire viral RNA genome and adapting them to avoid the possibility of false negatives.

When they tested for accuracy at different sample dilutions, they found that the assay was 100 percent accurate for samples with 200 copies per microliter over 30 minutes of measurement, with accuracy dropping to 50 percent at 50 copies per microliter.

"I think one aspect of the testing currently — especially for surveillance testing — that we all wish we had at the moment is that we could test ourselves every day," Ott said. "[What if every day,] you get up, you brush your teeth, and you take your test in the morning? And if you could do this every day, you would have a very good dynamic curve if you had indeed a positive test at one point." In this scenario, if at some point a person were to test positive, they would be able to isolate themselves immediately and limit their exposure to other people.

"The second aspect is that if you continue to use your measurements, you would have an idea when you reach a peak with your viral load, and when you go down," Ott noted. "And this dynamic aspect is something that we think is valuable."

The researchers also tested a reader device with a smartphone attached, in order to compare its ease of use and readability to conventional laboratory microscopes. Interestingly, they found that their device was approximately an order of magnitude more sensitive than the plate reader used in the development of the assay because measurement noise was reduced and they were able to collect data from additional time points, which decreased the uncertainty in their quantitative viral readings.

The device is still in the prototype stage, according to Ott. At base, it would require an optical element and a small heating device inside to release the viral RNA from the sample. The researchers are still determining the best way to integrate the smartphone. The actual reagents for the assay would be contained on a chip or cartridge — the user would take a nasal swab, introduce the sample to the chip, and insert the chip into the reader device where the reaction would take place. After the requisite time, the result would be displayed on the phone screen. The chip would be the only consumable for the user to deal with.

"You would not read the two bars like in a pregnancy test, but you would read it on your phone. And the phone would also basically be the processor, but also the connection to the cloud where you can store the data, where you can analyze the data remotely, and where, in the future, you could think about including [the data] in geo-surveillance with a GPS location of your phone," Ott said.

Ultimately, Ott and her colleagues are envisioning the test as an in-home assay, but she acknowledged that such assays can be difficult to get through the US Food and Drug Administration, especially when the use of a smartphone is involved.

"The at-home testing is a huge high bar because really nothing can go wrong and the consumer is supposed to [have] really minimal involvement," she said. "So, I think our first step is to aim to get this out and manufacture it as more of potentially a box at point of care, at the doctor's office, or the school, or the airport, where there's still [at least] minimally trained personnel involved that can handle this. But I think the longer-term vision is really that you would have something that you can clip on to any cell phone and can use at home."

There's still a lot of work to be done on both the box and the cartridge design, but Ott believes they can get the price of the cartridge to less than $10, depending on who the researchers potentially partner with to mass-produce the tests, if the assay does receive FDA approval. She noted that the team is engaged in conversations with potential partners but couldn't give additional details.

The importance of saliva

For Tony Hu, lead author of the Science Advances paper and a principle investigator at Tulane, the use of saliva as the sample type in the assay he and his colleagues developed is as important as the technology underpinning it. Nasopharyngeal swabs are not only unpleasant for patients as a sample collection method, but they've been found to be inconsistent in the amount of virus they can collect depending on the sensitivity of the patient, the experience of the healthcare worker collecting the sample, or the stage of the infection, he noted. This can lead to inaccurate test results. Saliva samples are much easier to give and to collect.

In their study, the researchers said they found that saliva and nasal samples from COVID-19-positive patients exhibit high positive-percentage agreement when analyzed within seven days of COVID-19 symptoms onset, with an indication that saliva samples may remain positive longer.

Given the uncertainty involved in human exposure events, it isn't possible to evaluate the relative diagnostic utility of saliva and nasopharyngeal swab samples early in infection, so the researchers used a rhesus macaque model, analyzing nasal and oropharyngeal swabs from seven macaques before and after SARS-CoV-2 infection. Their assay determined that mean SARS-CoV-2 RNA levels were 3.6-fold to 124-fold higher, and more stable, in oropharyngeal versus nasal swab samples at all times points after infection, suggesting that the usability of saliva as a robust diagnostic sample for both early and later infection is worth exploring.

Similarly to Ott and her colleagues, Hu, Ning, and their collaborators are envisioning their test primarily for home use.

"It's necessary for all people to have the capability of self-testing," Hu said. "When the pandemic started in China and the hospitals were so crowded, they calculated there were 16 percent of patients that really carried the virus. Another 40 percent of patients probably had other pathogens like the flu and they were infected [with SARS-CoV-2] in the hospitals there."

Now, he said, people have become slightly paranoid and they become nervous that they may have COVID-19 when they cough or have a fever. A reliable home test would allow them to check first to see if they are indeed infected with SARS-CoV-2 before going to the hospital, or if they merely have a cold or the flu.

The researchers are designing a reader device that would include a dock for the sample chip, some kind of heating element to release the viral RNA, and a fluorescence filter that would enable the cell phone camera to read the signal. With this filter, any cell phone camera would be able to detect the signal from the assay, Ning said.

"The top priority is the self-use for all people who have the capability," he said. "If you have a cell phone, the only thing probably you need to have is [the box] we'll design, and all the chips that we're making can handle multiple samples — it's for family use. And of course, if we can serve the airports, the train stations, or restaurants, that would that would be fantastic. And that's probably the next target."

Ning is also working on a multiplex version of the test chip that would allow it to detect multiple pathogens at once. He has some preliminary data on the detection of influenza A and B, and said those could easily be integrated into the same chip as SARS-CoV-2.

The fundamental CRISPR assay technology has been licensed by a company called Nanopin Technologies, Hu said. The cellphone-based detector hasn't been licensed yet, but he said he's received several emails from various firms interested in possible commercialization deals.

"Of course, we would like to commercialize it, and as quickly as possible," Hu said.

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