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EDITORS' PICK | Dec 13, 2020, 06:39pm EST | 2,227 views

Can Crispr-Based Covid-19 Testing Using Smartphones Slow

The Pandemic?
Robert Gla er, MD Contributor
Healthcare
I cover breaking news in medicine, med tech and public health

A new CRISPR-based test for COVID-19 developed by... [+]

GLADSTONE INSTITUTES

We continue to struggle with the ongoing pandemic in the U.S. due to political and
cultural influences on mitigation measures (wearing masks, distancing), the lack of a
comprehensive national testing strategy, but also due to the difficulty in reliably
identifying symptomatic, asymptomatic and pre-symptomatic carriers of SARS-CoV-2,
the virus responsible for Covid-19.

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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.

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.

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How Smartphone-Based COVID-19 Test Gives Results In 30 Min…

Min…

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.
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“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.

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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.

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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
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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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“These assays that use Crispr as well as tests like Jonathan Rothberg’s hybrid
LAMP/paper strip test (the proteins on the paper strip capture the amplicons, instead of
unamplified antigen) can be very very powerful,” added Mina.

Paradigm for Covid-19 Screening and Surveillance

The current gold standard approach for diagnosing acute infection with SARS-Cov-2,
the PCR assay, is inappropriately and widely used as an approach for general screening
in the U.S.
Issues with PCR include prolonged turnaround time (still several days, in some cases)—
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allowing one to continue transmitting the virus while awaiting results if not
quarantined—as well as it’s high sensitivity, which can be problematic in those people
who end up testing positive after the period of acute infection has passed, as small
fragments of non-infectious RNA remain in the body turning the test “positive”,
although you are not infectious to others and no longer able to spread the disease. This
results in needless quarantines of non-infectious individuals from falsely positive
results, but also the potential for false negatives if performed at points before or after
the the viral load peaks. Instead, PCR’s are ideal for someone who is acutely ill with the
virus, for whom you have high suspicion of illness.

Consequently, a PCR test’s utility in screening and surveillance is compromised by both

lack of speed and being too sensitive a test for the purpose of conducting surveillance
and reducing transmission in an at-risk population. Instead, what’s actually needed are
point-of-care tests which have a lower sensitivity, but can be performed in the home
setting (like a pregnancy test) more often in order to make up for a lower threshold of
detection. This is the premise behind rapid viral antigen testing which detects small of
viral proteins instead of viral RNA fragments.

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While there may certainly be enthusiasm for a Crispr-based smartphone approach for
testing, Mina believes that “they will not be able to scale as quickly as high-volume
antigen tests at this point, so I think a terrific use of them could be the orthogonal
confirmation of rapid antigen tests. These rapid molecular tests can serve as the gold
standard in many ways. And I believe that they serve to indicate a changing tide in
medical diagnostics and public health, a democratization of testing which people
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clearly are yearning for.”

But one important issue with tests that are so sensitive is detecting viral RNA fragments
in those who aren’t infectious any longer, testing positive— but not able to spread
Covid-19. “The only lingering issue for molecular RNA SARS-C0V-2 detecting tests is that
they are so sensitive that like PCR, they will detect RNA long after somebody has been
infected and infectious,” explained Mina. “This has been a major issue that has not
been well discussed by the regulatory agencies nor the CDC but it is a problem.”

“People can remain positive for months potentially after being infectious—that’s why
these tests have to be used cautiously, he warned. “If they are used as confirmatory test
for the rapid antigen tests, which can sometimes have small numbers of false positives,
then they become very very powerful confirmatory tools because they are identifying
entirely distinct molecules. In addition, if they are being used after a positive rapid
antigen test, the likelihood that somebody who turns positive on the RNA test who is
still infectious is very high, so using it to confirm rapid antigen tests (which are positive
only when someone is likely infectious) sort of solves the issue of detecting people long
after they have been infectious.” 

“But the molecular tests that can be used at home hold so much more promise as well
for the future of infectious disease monitoring and many other conditions: they should
become part of our normal lives in the future so that people do not have to always go
visit a doctor if they just want to know if they have influenza or if their child has other
viruses,” offered Mina.

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“They can also serve to provide a powerful tool for many other diagnostic uses in the
future,” he concluded.

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Robert Gla er, MD

I am an emergency physician on staff at Lenox Hill Hospital in New York City, where I have
practiced for the past 15 years. I also serve as an adviser and editor to… Read More

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