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Imagine dabbing your nostrils, inserting the swab into a device, and getting a reading on your phone in 15-30 minutes telling you if you are infected with the COVID-19 virus. This was the vision for a team of scientists at Gladstone Institutes, University of California, Berkeley (UC Berkeley) and University of California, San Francisco (UCSF). And now, they report a scientific breakthrough that brings them closer to turning this vision into reality.
One of the main obstacles to the fight against the COVID-19 pandemic and the complete reopening of communities across the country is the availability of mass rapid tests. Knowing who is infected would provide valuable information on the potential spread and threat of the virus for both politicians and citizens.
However, people often have to wait several days for their results, or even longer when there is a backlog in processing laboratory tests. And the situation is aggravated by the fact that most of the infected people have mild or no symptoms, but are still carriers and spread of the virus.
In a new study published in the scientific journal Cell, the team from Gladstone, UC Berkeley, and UCSF outlined the technology for a CRISPR-based test for COVID-19 that uses a smartphone camera to deliver accurate results in less than 30 minutes.
“It has been an urgent task for the scientific community to not only ramp up testing but also provide new testing options,” says Melanie Ott, MD, PhD, director of the Gladstone Institute of Virology and one of the study leaders. “The test we developed could provide rapid, low-cost tests to help control the spread of COVID-19.”
The technique was designed in collaboration with UC Berkeley bioengineer Daniel Fletcher, PhD, as well as Jennifer Doudna, PhD, who is a senior researcher at Gladstone, a professor at UC Berkeley, president of the Innovative Genomics Institute, and an investigator of the Howard Hughes Medical Institute. Doudna recently won the 2020 Nobel Prize in Chemistry for discovering CRISPR-Cas genome editing together, the technology behind this work.
Their new diagnostic test can not only generate a positive or negative result, but it also measures the viral load (or concentration of SARS-CoV-2, the virus that causes COVID-19) in a given sample.
“When coupled with repeated testing, viral load measurement could help determine whether an infection is increasing or decreasing,” says Fletcher, who is also a Chan Zuckerberg Biohub Investigator. “Monitoring the course of a patient’s infection could help healthcare professionals estimate the stage of infection and predict, in real time, how much time is likely to be required for recovery.”
A simpler test thanks to direct detection
Current COVID-19 tests use a method called quantitative PCR, the gold standard of testing. However, one of the problems with using this technique to test for SARS-CoV-2 is that it requires DNA. Coronavirus is an RNA virus, which means that to use the PCR approach, viral RNA must first be converted into DNA. Additionally, this technique relies on a two-step chemical reaction, including an amplification step to provide enough DNA to make it detectable. Therefore, current tests typically require skilled users, specialized reagents, and cumbersome laboratory equipment, which severely limits where tests can be performed and causes delays in receiving results.
As an alternative to PCR, scientists are developing testing strategies based on CRISPR gene editing technology, which excels in specific identification of genetic material.
All CRISPR diagnostics to date have required viral RNA to be converted to DNA and amplified before it could be detected, adding time and complexity. In contrast, the new approach described in this recent study skips all conversion and amplification steps, using CRISPR to directly detect viral RNA.
“One of the reasons we’re excited about CRISPR-based diagnostics is the potential for fast and accurate results when needed,” says Doudna. “This is especially useful in places with limited access to testing, or when rapid and frequent testing is needed. It could eliminate many of the bottlenecks we’ve seen with COVID-19.”
Parinaz Fozouni, a UCSF graduate student working in Ott’s lab in Gladstone, has been working on an RNA detection system for HIV in recent years. But in January 2020, when it became clear that coronavirus was becoming a bigger problem globally and that testing was a potential trap, she and her colleagues decided to shift their focus to COVID-19.
“We knew that the test we were developing would make sense to help the crisis by enabling rapid tests with minimal resources,” says Fozouni, co-first author of the article, along with Sungmin Son and María Díaz de León Derby of The Fletcher team at UC Berkeley. “Instead of the well-known CRISPR protein called Cas9, which recognizes and cleaves DNA, we used Cas13, which cleaves RNA.”
In the new test, the Cas13 protein is combined with a reporter molecule that fluoresces when cut and then mixed with a patient sample from a nasal swab. The sample is placed in a device that connects to a smartphone. If the sample contains RNA from SARS-CoV-2, Cas13 will be activated and will cut the reporter molecule, resulting in the emission of a fluorescent signal. Then, the smartphone camera, essentially converted into a microscope, can detect fluorescence and report that a swab has tested positive for the virus.
“What makes this test truly unique is that it uses a one-step reaction to directly test viral RNA, as opposed to the two-step process in traditional PCR tests,” says Ott, who is also a professor in the Department of Medicine at UCSF. “The simpler chemistry, paired with the smartphone camera, reduces detection times and does not require complex laboratory equipment. It also allows the test to produce quantitative measurements rather than simply a positive or negative result.”
The researchers also say their test could be adapted to a variety of cell phones, making the technology easily accessible.
“We chose to use cell phones as the basis for our detection device as they have intuitive user interfaces and highly sensitive cameras that we can use to detect fluorescence,” explains Fletcher. “Cell phones are also mass-produced and cost-effective, demonstrating that no specialized lab tools are needed for this test.”
Fast and accurate results to limit the pandemic
When the scientists tested their device using patient samples, they confirmed that it could provide a very fast response time of results for samples with clinically relevant viral loads. In fact, the device accurately detected a series of positive samples in less than 5 minutes. For samples with a low viral load, the device took up to 30 minutes to distinguish it from a negative test.
“Recent SARS-CoV-2 models suggest that frequent testing with fast response times is what we need to overcome the current pandemic,” says Ott. “We hope that with more testing, we can avoid blockages. and protect the most vulnerable populations “.
Not only does the new CRISPR-based test offer a promising option for rapid testing, but by using a smartphone and avoiding the need for bulky lab equipment, it has the potential to go portable and eventually be made available for point-of-care or even at home. And it could also be expanded to diagnose other respiratory viruses besides SARS-CoV-2.
Additionally, the high sensitivity of smartphone cameras, coupled with their connectivity, GPS, and data processing capabilities, have made them attractive tools for diagnosing diseases in low-resource regions.
“We hope to develop our test into a device that can instantly upload results to cloud-based systems while maintaining patient privacy, which would be important for contact tracing and epidemiological studies,” says Ott. “This type of Smartphone-based diagnostic testing could play a crucial role in controlling current and future pandemics. “
Information on the research project
The study titled “Amplification-free Detection of SARS-CoV-2 with CRISPR-Cas13a and Cell Phone Microscopy”, was published online by Cell on December 4, 2020.
Other authors on the study include Gavin J. Knott, Michael V. D’Ambrosio, Abdul Bhuiya, Max Armstrong, and Andrew Harris of UC Berkeley; Carley N. Gray, G. Renuka Kumar, Stephanie I. Stephens, Daniela Boehm, Chia-Lin Tsou, Jeffrey Shu, Jeannette M. Osterloh, Anke Meyer-Franke and Katherine S. Pollard of the Gladstone Institutes; Chunyu Zhao, Emily D. Crawford, Andreas S. Puschnick, Maira Phelps and Amy Kistler of Chan Zuckerberg Biohub; Neil A. Switz of San Jose State University; and Charles Langelier and Joseph L. DeRisi of UCSF.
The research was supported by the National Institutes of Health (NIAID grant 5R61AI140465-03 and NIDA grant 1R61DA048444-01); the NIH Rapid Acceleration of Diagnostics (RADx) program; the National Heart, Lung, and Blood Institute; the National Institute of Biomedical Imaging and Bioengineering; the Department of Health and Human Services (Grant No. 3U54HL143541-02S1); as well as through the philanthropic support of Fast Grants, the James B. Pendleton Charitable Trust, the Roddenberry Foundation and multiple individual donors. This work was also made possible by a generous gift from an anonymous private donor in support of the ANCeR diagnostics consortium.
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