Imagine dabbing your nostrils, sticking the swab in a device, and in 15 to 30 minutes you will get an indication on your phone telling you if you are infected with the COVID-19 Virus. This was the vision of a team of scientists at the Gladstone Institutes. University of California, Berkeley (UC Berkeley) and University of California, San Francisco (UCSF). And now they report a scientific breakthrough that will bring them closer to realizing that vision.
One of the biggest hurdles in fighting the COVID-19 pandemic and fully reopening communities across the country is the availability of rapid mass tests. Knowing who is infected would provide valuable insights into the potential spread and threats of the virus for both policy makers and citizens.
However, people often have to wait several days for their results, or even longer if there is a backlog in processing laboratory tests. The situation is made worse by the fact that most infected people have mild or no symptoms and still carry and spread the virus.
Published in a new study in the journal cellThe team from Gladstone, UC Berkeley and UCSF has presented the technology for a CRISPR-based test for COVID-19, which uses a smartphone camera to achieve accurate results in less than 30 minutes.
“It was an urgent task for the scientific community not only to improve the tests, but also to provide new test options,” says Dr. Melanie Ott, director of the Gladstone Institute of Virology and one of the leaders of the study. “The assay we have developed could enable quick and inexpensive tests to control the spread of COVID-19.”
The technique was developed in collaboration with UC Berkeley bioengineer Daniel Fletcher and Jennifer Doudna, PhD, Senior Investigator at Gladstone, Professor at UC Berkeley, President of the Innovative Genomics Institute, and Investigator at the Howard Hughes Medical Institute. Doudna recently received the 2020 Nobel Prize in Chemistry for jointly discovering CRISPR-Cas genome editing, the technology behind this work.
In addition to giving a positive or negative result, your new diagnostic test can measure viral load (or the concentration of) SARS-CoV-2, the virus that causes COVID-19) in a specific sample.
“In conjunction with repeated testing, measuring viral load can help determine whether an infection is increasing or decreasing,” says Fletcher, who is also a Chan Zuckerberg Biohub Investigator. “Monitoring the progress of a patient’s infection could help health professionals assess the stage of the infection and predict in real time how long recovery is likely to take.”
A simpler test through direct detection
Current COVID-19 tests use a method called quantitative PCR – the gold standard for testing. However, one of the problems with using this technique to test for SARS-CoV-2 is that it is required DNA. Coronavirus is a RNA Virus, which means that in order to use the PCR approach, the viral RNA must first be converted into DNA. In addition, this technique relies on a two-step chemical reaction, including an amplification step, to provide enough DNA to make it detectable. Current tests therefore typically require trained users, special reagents, and cumbersome laboratory equipment, which severely limit testing options and lead to delays in receiving results.
As an alternative to PCR, scientists are developing test strategies based on the CRISPR gene editing technology, which is characterized by the specific identification of genetic material.
In all previous CRISPR diagnoses, the viral RNA had to be converted to DNA and amplified before it could be detected, adding time and complexity. In contrast, the novel approach described in this recent study skips all conversion and amplification steps and uses CRISPR to detect the viral RNA directly.
“One reason we are excited about CRISPR-based diagnostics is the potential for quick and accurate results where they are needed,” says Doudna. “This is especially useful in places with limited access to testing or when frequent, rapid testing is required. It could remove many of the bottlenecks we saw with COVID-19. ”
Parinaz Fozouni, a UCSF PhD student who works in Ott’s laboratory in Gladstone, had been working on an RNA detection system for HIV for the past several years. When it became clear in January 2020 that coronavirus was becoming a bigger problem around the world and testing was a potential threat, she and her colleagues decided to shift their focus to COVID-19.
“We knew that the test we developed was logically suitable for dealing with the crisis by enabling rapid testing with minimal resources,” says Fozouni, co-first author of the paper, along with Sungmin Son and María Díaz de León Derby from Fletcher’s team at UC Berkeley. “Instead of the well-known CRISPR protein 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, which fluoresces when cut, and then mixed with a patient sample from a nasal swab. The sample is placed in a device that is connected to a smartphone. If the sample contains RNA from SARS-CoV-2, Cas13 is activated and cuts the reporter molecule, emitting a fluorescent signal. Then the smartphone camera, essentially converted into a microscope, can capture the fluorescence and report that a swab has tested positive for the virus.
“What makes this test really unique is that it tests the viral RNA directly in one step, as opposed to the two-step process in conventional PCR tests,” says Ott, who is also a professor in the Department of Medicine at UCSF. “The simpler chemistry in connection with the smartphone camera shortens the detection time and does not require complex laboratory equipment. In addition, the test can provide quantitative measurements, rather than just a positive or negative result. ”
The researchers also say their assay could be adapted to a wide variety of cell phones, making the technology easily accessible.
“We decided to use cell phones as the basis for our detection device because 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 inexpensive. This shows that no special laboratory instruments are required for this test.”
Accurate and quick results to limit the pandemic
When the scientists tested their device on patient samples, they confirmed that it can provide a very fast turnaround time for results on samples with clinically relevant viral load. In fact, the device accurately captured a set 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.
“Newer models of SARS-CoV-2 suggest that frequent tests with a fast turnaround time are required to overcome the current pandemic,” says Ott. “We hope that as testing increases, we can avoid bans and protect the most vulnerable populations.”
Not only does the new CRISPR-based test offer a promising option for quick testing, but by using a smartphone and avoiding bulky lab equipment, it can also be made portable and ultimately made available for point-of-care operations or at home . It could also be expanded to diagnose other respiratory viruses beyond SARS-CoV-2.
In addition, the high sensitivity of smartphone cameras, along with their connectivity, GPS, and data processing capabilities have made them attractive tools for diagnosing diseases in regions with limited resources.
“We hope to develop our test into a device that enables results to be uploaded instantly to cloud-based systems while protecting patient privacy. This would be important for contact tracing and epidemiological studies, ”says Ott. “These types of smartphone-based diagnostic tests could play a vital role in controlling current and future pandemics.”
Reference: “Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy” by Parinaz Fozouni, Sungmin son, Maria Díaz de León Derby, Gavin J. Knott, Carley N. Gray, Michael V. D ‘Ambrosio, Chunyu Zhao, Neil A. Switz, G. Renuka Kumar, Stephanie I. Stephens, Daniela Böhm, Chia-Lin Tsou, Jeffrey Shu, Abdul Bhuiya, Max Armstrong, Andrew R. Harris, Pei-Yi Chen, Jeannette M. Osterloh, Anke Meyer-Franke, Bastian Joehnk, Keith Walcott, Anita Sil, Charles Langelier, Katherine S. Pollard, Emily D. Crawford, Andreas S. Puschnik, Maira Phelps, Amy Kistler, Joseph L. DeRisi, Jennifer A. Doudna, Daniel A. Fletcher and Melanie Ott, December 4, 2020, cell.
DOI: 10.1016 / j.cell.2020.12.001
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 Böhm, Chia-Lin Tsou, Jeffrey Shu, Jeannette M. Osterloh, Anke Meyer-Franke and Katherine S. Pollard from the Gladstone Institutes; Chunyu Zhao, Emily D. Crawford, Andreas S. Puschnick, Maira Phelps and Amy Kistler from the 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 program for the rapid acceleration of diagnosis (RADx); the National Institute of Heart, Lungs and Blood; the National Institute for Biomedical Imaging and Bioengineering; the Department of Health and Human Services (Grant No. 3U54HL143541-02S1); as well as through philanthropic support from Fast Grants, the James B. Pendleton Charitable Trust, the Roddenberry Foundation, and several individual donors. This work was also made possible by a generous gift from an anonymous private donor to support the ANCeR Diagnostic Consortium.