Can Crispr-Based Covid-19 Testing Using Smartphones Slow The Pandemic?

Reposted from this article (https://www.forbes.com/sites/robertglatter/2020/12/13/can-crispr-based-covid-19-testing-using-smartphones-slow-the-pandemic/amp/?__twitter_impression=true)

“While we typically wait for many days to get results from PCR-based nasal swabs, a test which has been inappropriately used to screen the U.S. population for SARS-CoV-2, two recent proof-of-concept studies using novel smartphone-based Crispr technology coupled with optics and fluorescence detection may be poised to change how we approach not only rapid testing and screening, but also testing for acute infection. Interestingly, the motivation for research to enable Crispr-based Covid-19 monitoring was actually inspired by the need for rapid HIV testing and for patients to be aware of their viral loads without the need for formal laboratory monitoring.
Newly published research in the journal Cell last week from Gladstone Institutes, University of California, San Francisco and University of California, Berkeley sets the stage for such a shift in our current paradigm for testing. Dr. Jennifer Doudna, one of the collaborating authors of this novel amplification-free RNA approach to Crispr-based smartphone testing described in this paper, was also the co-recipient (along with Emmanuelle Carpentier) of the 2020 Nobel prize in Chemistry for her pioneering work in Crispr gene editing.

In short, Crispr has the potential to augment our current approach to testing if, in future studies, it is shown to demonstrate comparable or improved accuracy or sensitivity (as the gold standard PCR-based approach). This novel technology is now possible due to advances that obviate the need to amplify SARS-CoV-2 RNA, a rate-limiting step in standard Crispr technology, thus enabling point-of-care testing using smartphones.

Our study shows that we can do the detection part of this assay very quickly, making the measurement with mass-produced consumer electronics,” said Daniel Fletcher, PhD, a bioengineer at the University of California in Berkeley and co-senior author on the paper, in a press release. “We don’t need fancy laboratory equipment.”

To be more specific, the researchers developed a proof-of-concept amplification-free, rapid Crispr-Cas13a assay to directly detect SARS-CoV-2 from nasal swab RNA that can be read by converting a cellphone camera into a specialized microscope. This assay, distinct from prior Crispr diagnostics, does not require pre-amplification of the viral genome (utilizing DNA) to ultimately detect SARS CoV-2 RNA. Specifically, RNA in the sample can be detected using the Cas13 enzyme, eliminating the need for reverse transcription of RNA into DNA, and then amplification by PCR-based technology used in current standard tests. Moreover, by employing direct detection of the viral RNA, the test is able to provide a numerical value of RNA copies, as opposed to just a positive or negative result, as with standard PCR-based testing. The new assay is simple, portable and measures fluorescence using a smartphone camera that attaches to a compact laser-based device.

“It’s super exciting to have this quantitative aspect in the assay,” said Melanie Ott, MD, PhD, a virologist at Gladstone Institutes and the University of California, San Francisco. “PCR is the gold standard, but you have to go through so many steps. There are huge opportunities here for pathogens and for biology in general to make RNA quantification more precise.”

The clinical value of this novel amplification-free Crispr-based approach certainly lies in its speed, without compromising sensitivity. The assay correctly identified all SARS-CoV-2 positive RNA samples from patients tested [Cycle threshold (Ct) values 14-22] within 5 minutes after being measured on the smartphone device, demonstrating its potential value as a rapid, accurate, portable and potentially low-cost approach for point-of-care SARS-CoV-2 screening. Obviously, additional work will be required to translate this proof-of-concept study into widely available point-of-care device.

While nasal swab RNA was used for evaluation in Ott and Fletcher’s study, another group of researchers from Tulane led by Drs. Bo Ning and Tony Hu decided to pursue the potential for a 15 minute saliva-based point-of-care test, also employing the use of Crispr and smartphone-based technology. Their proof of concept study, published last week in Science Advances, tested in 12 people with active Covid-19 infections as well as 6 healthy controls.

In an nutshell, the Tulane researchers found that their approach, which combines a fluorescence microscope readout device with a smartphone to determine viral load from a Crispr/Cas12a assay, worked as effectively as the well-established quantitative PCR method.

“We believe this smartphone platform, a similar future application, offers the potential to rapidly expand Covid-19 screening capacity, and potentially simplify the verification of contact tracing, to improve local containment and inform regional disease control efforts,” the authors write.

At this time, Covid-19 testing requires swabbing the upper part of the throat behind the nose. It’s an uncomfortable process that requires medical professionals in full PPE to collect samples before running PCR tests. However, recent studies have found that SARS-CoV-2 may be equally present in the nasopharynx and the saliva during early infection, suggesting saliva-based Covid-19 tests could enable comparably reliable but simpler, safer testing. And while saliva may be less sensitive compared to nasal swab RNA for PCR-based testing, this may still be acceptable if frequently used for screening or surveillance in those who remain asymptomatic.

To develop a widely accessible platform for saliva-based testing, Ning and colleagues built a prototype assay chip that uses the Crispr/Cas12a enzyme to enhance an amplified viral RNA target’s signal within a saliva sample. They integrated the chip into a smartphone-based fluorescence microscope readout device, which captures and analyzes images to determine whether the virus is present above a threshold concentration. The researchers using this approach successfully distinguished between patients with and without the virus.

The Tulane researchers further compared nasal and saliva swabs from non-human primates before and after infection with Covid-19. They found higher SARS-CoV-2 RNA levels in saliva, further suggesting that saliva may provide a promising means of diagnosis, and potential surveillance, after infection. They also believe that a future version of the chip used in this technique could contain pre-loaded reagents and sample controls, paired with a smartphone app could transmit data securely, thereby strengthening the power of telehealth to allow contact tracing and population surveillance during the pandemic.

Eric Topol, M.D, Executive VP, Scripps Research, Professor, Molecular Medicine, Scripps Research, Director & Founder, Scripps Research Translational Institute, and Editor-in-Chief of Medscape feels that development of such technologies will accelerate progress in developing rapid, home-based Covid-19 testing. “I do believe this remarkable capability of using smartphone, Crispr genome editing, a kit, and a swab (preferably from anterior nares, not saliva) will enable very rapid, accurate, and inexpensive home tests for Covid-19 infectiousness. Having these or other rapid home tests widely and freely available ASAP—in every household—is essential to contain the pandemic.”

Michael Mina MD, PhD, Assistant Professor of Epidemiology at the Harvard T.H. Chan School of Public Health, Assistant Professor in Immunology and Infectious Diseases at the Harvard Chan School and Associate Medical Director in Clinical Microbiology and Virology in the Department of Pathology at Brigham and Women’s Hospital, Harvard Medical School, concurred. “I think these [CRISPR-based] approaches hold tremendous promise! Theoretically, they can become as sensitive and specific as PCR—and potentially more specific than lab-based PCR because there can be less chance of contamination from other specimens.”

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