Sunday , March 7 2021

New COVID-19 Test Uses CRISPR Smart Phone Camera and Genetic Technology



CRISPR Based COVID Test Using a Smartphone

A new CRISPR-based test for COVID-19 developed by researchers at Gladstone Institutes, UC Berkeley, and UC San Francisco essentially converts a smartphone camera into a microscope to provide fast and accurate results. Credit: Gladstone Institutions

Imagine swabbing your nostrils, putting the swab in a device, and having a reading out on your phone in 15 to 30 minutes that tells you if you’re infected with the COVID-19 virus. This has been the vision for a team of scientists at the Gladstone Institutions, University of California, Berkeley (UC Berkeley), and the University of California, San Francisco (UCSF). And now, they report a scientific breakthrough that brings them closer to making this vision a reality.

One of the major obstacles to combating COVID-19 pandemic communities and full reopening nationwide is the availability of mass rapid tests. Knowing who is infected would provide valuable insights into the spread and potential threat of the virus to policy makers and citizens alike.

Yet 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 exacerbated by the fact that most infected people have mild or no symptoms, yet they carry and spread the virus.

In a new study published in the scientific journal Cell, the team from Gladstone, UC Berkeley, and UCSF have 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.

Melanie Ott and Parinaz Fozouni

A team of scientists from Gladstone, UC Berkeley, and UC San Francisco, including Melanie Ott (left) and Parinaz Fozouni (right), outlined the technology for rapid, one-step mobile testing that could help combat the pandemic and fully reopened. . Credit: Gladstone Institutions

“It has been an urgent task for the scientific community to not only increase testing, but also provide new testing options,” said Melanie Ott, MD, PhD, director of the Gladstone virology Institute and one of the study’s leaders. “The assay we developed could provide fast, low-cost tests to help control the spread of COVID-19.”

The technique was designed in collaboration with UC Berkeley bioenergist Daniel Fletcher, PhD, as well as Jennifer Doudna, PhD, senior researcher at Gladstone, a professor at UC Berkeley, president of the Institute for Innovative Genomics, and researcher the Howard Hughes Medical Institute. Doudna recently won the 2020 Nobel Prize in Chemistry for co-discovering CRISPR-Cas genome editing, the technology that underpins this work.

Not only can their new diagnostic test produce a positive or negative result, it also measures the viral load (or concentration) SARS-CoV-2, the virus that causes COVID-19) in a given sample.

“Combined with repeated testing, measuring viral load could help determine whether an infection increases or decreases,” said Fletcher, who is also a Chan Zuckerberg Biohub Researcher. “Monitoring a patient’s course of infection could help healthcare professionals estimate the stage of infection and predict, in real time, how long it is likely to cure.”

Simplified Testing by Direct Detection

Current COVID-19 tests use a method called quantitative PCR – the gold standard of tests. However, one of the problems with using this technique to test for SARS-CoV-2 is that it is required DNA. A coronavirus is a RNA virus, which means that to use the PCR method, 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 of the DNA to make it detectable. Therefore, current tests typically require trained users, specialist reagents, and onerous laboratory equipment, which severely limits where tests can take place and delays in receiving results.

As an alternative to PCR, scientists are developing testing strategies based on the CRISPR gene editing technology, which exceeds the specific identification of genetic material.

All CRISPR diagnostics have so far required the viral RNA to be converted into DNA and amplified before it can be detected, adding time and complexity. In contrast, the new method described in this recent study skips all conversion and amplification steps, using CRISPR to directly detect the viral RNA.

“One reason we’re excited about CRISPR-based diagnostics is the potential for fast, accurate results on need,” said Doudna. “This is especially useful in places with limited access to tests, or where rapid tests are often needed. It could eliminate a lot of the congestion we’ve seen with COVID-19. “

COVID-19 Test Smart Phone Camera

Fozouni (left), a graduate student in Ott’s lab (right), is the first co-author of the study published in Cell. Credit: Gladstone Institutions

Parinaz Fozouni, a UCSF graduate student working at Ott’s lab in Gladstone, had been working on an RNA sensing system for HIV for the past few years. But in January 2020, when it became clear that the coronavirus was becoming a bigger issue globally and testing was a potential problem, she and her colleagues decided to shift their focus to COVID-19.

“We knew that the assay we were developing would be a logical fit to help the crisis by allowing quick tests with little resources,” said Fozouni, the first co-author of the paper, along with Sungmin Son and María Díaz de León Derby of Team Fletcher at UC Berkeley. “Instead of the well-known CRISPR protein called Cas9, which recognizes and clears DNA, we used Cas13, which clears RNA.”

In the new test, the Cas13 protein is combined with a reporter molecule that becomes fluorescent when cut, and then mixed with a patient sample of a nasal swab. The sample is placed in a device that is attached to a smartphone. If the sample contains RNA from SARS-CoV-2, Cas13 will be activated and will break down the reporter molecule, causing the emission of a fluorescent signal. The smartphone camera, which is basically converted into a microscope, can then detect the fluorescence and report that a swab has tested positive for the virus.

“What really makes this test unique is that it uses a one-step reaction to directly test the viral RNA, rather than the two-step process in traditional PCR tests,” said Ott, who is a professor in the Department of Medicine. at UCSF. “The simpler chemistry, paired with the smartphone camera, reduces detection time and does not require complicated lab equipment. It also allows the test to produce quantitative measurements rather than just a positive or negative result. “

The researchers also say that their assay could be adapted to a variety of mobile phones, making the technology accessible.

“We chose to use mobile phones as the basis for our sensing device as they have intuitive user interfaces and very sensitive cameras that we can use to detect fluorescence,” explained Fletcher. “Mobile phones are also mass-produced and cost-effective, demonstrating that specialized lab instruments are not required for this assay.”

Accurate and Quick Results to Limit the Pandemic

When the scientists tested their device using patient samples, they confirmed that it could provide a very fast turnaround time of results for samples with clinically relevant viral loads. In fact, the device correctly detected a set of positive samples in less than 5 minutes. For samples with a low viral load, the device required up to 30 minutes to distinguish it from a negative test.

“Recent models of SARS-CoV-2 suggest that testing often with fast turnaround time is what we need to overcome the current pandemic,” said Ott. “We hope that with more testing we can avoid locking up and protecting the most vulnerable populations.”

Not only does the new CRISPR-based test offer a promising option for rapid tests, but by using a smartphone and avoiding the need for bulky lab equipment, it has the potential to become portable and portable it will eventually be available for point of care or even home use. And it could also be expanded to diagnose other respiratory viruses beyond SARS-CoV-2.

In addition, the high sensitivity of smartphone cameras, together with their connectivity, GPS, and data processing capabilities, have made them attractive tools for diagnosing disease in low resource regions.

“We hope to develop our test into a device that could upload instant results to cloud systems while maintaining patient privacy, which would be important for contact tracking and epidemiologic studies,” said Ott. “This type of smartphone-based diagnostic test could play a vital role in managing the current and future pandemics.”

Reference: “SARS-CoV-2 detection without amplification with CRISPR-Cas13a and mobile phone microscopy” by Parinaz Fozouni, Sungmin Son, María 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 Boehm, 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, 4 December 2020, Cell.
DOI: 10.1016 / j.cell.2020.12.001

Other authors of 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 Gladstone Institutes; Chunyu Zhao, Emily D. Crawford, Andreas S. Puschnick, Maira Phelps, and Amy Kistler of 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 Rapid Diagnostics Acceleration (RADx) program; 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 philanthropic support from Rapid Grants, the James B. Pendleton Charitable Trust, the Roddenberry Foundation, and multiple individual donors. This work was also made possible by a generous donation from an anonymous private donor to support the ANCeR diagnostics consortium.




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