Researchers Develop Novel COVID-19 Diagnostics Utilizing CRISPR Testing Platform And Smartphone Camera
: Researchers from Gladstone Institutes, University of California, Berkeley (UC Berkeley), and University of California, San Francisco (UCSF) have developed a new fast and portable yet accurate COVID-19 diagnostic platform utilizing CRISPR testing technology and the smartphone camera.
The SARS-CoV-2 coronavirus outbreak has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic and pre-symptomatic carriers of the virus.
CRISPR technology based testing can augment gold-standard PCR-based diagnostics if they can be made rapid, portable and accurate.
The study team reports the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ~100 copies/µL sensitivity in less than 30 minutes of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in less than 5 minutes.
The team combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity, and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARSCoV-2.
The research and development was published in the peer reviewed journal: Cell. https://www.cell.com/cell/pdf/S0092-8674(20)31623-8.pdf
The new developing would enable people to swab their nostrils, putting the swab in a device, and getting a read-out on their phone in 15 to 30 minutes that tells them if they are infected with the COVID-19 virus.
This has been the vision for the study research team and now, they report a scientific breakthrough that brings them closer to making this vision a reality.
To date, one of the key hurdles to combating the COVID-19 pandemic and fully reopening communities across the country is the availability of mass rapid testing. Knowing who is infected would provide valuable insights about the potential spread and threat of the virus for policymakers and citizens alike.
Presently individuals must often wait several days for their results, or even longer when there is a backlog in processing lab tests. And, the situation is worsened by the fact that most infected individuals have mild or no symptoms, yet still carry and spread the SARS-CopV-2 virus.
The team has outlined the technology for a CRISPR-based test for COVID-19 that uses a smartphone camera to provide accurate results in under 30 minutes.
Dr Melanie Ott, MD, Ph.D., director of the Gladstone Institute of Virology and one of the leaders of the study told Thailand Medical News, "It has been an urgent task for the scientific community to not only increase testing, but also to provide new testing options. The assay we developed could provide rapid, low-cost testing to help control the spread of COVID-19."
The new diagnostic platform was designed in collaboration with UC Berkeley bioengineer Dr Daniel Fletcher, Ph.D., as well as Dr Jennifer Doudna, Ph.D., who is a senior investigator a
t Gladstone, a professor at UC Berkeley, president of the Innovative Genomics Institute, and an investigator of the Howard Hughes Medical Institute.
Dr Doudna recently won the 2020 Nobel Prize in Chemistry for co-discovering CRISPR-Cas genome editing, the technology that underlies this work.
Importantly not only can their new diagnostic test generate a positive or negative result, it also measures the viral load (or the concentration of SARS-CoV-2, the virus that causes COVID-19) in a given sample.)
Dr Fletcher added, "When coupled with repeated testing, measuring viral load could help determine whether an infection is increasing or decreasing. Monitoring the course of a patient's infection could help health care professionals estimate the stage of infection and predict, in real time, how long is likely needed for recovery."
At present, COVID-19 tests use a method called quantitative PCR iethe gold standard of testing. However, one of the issues 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, the viral RNA must first be converted to 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.
Hence current tests typically need trained users, specialized reagents, and cumbersome lab equipment, which severely limits where testing can occur and causes delays in receiving results.
However, as an alternative to PCR, researchers are developing testing strategies based on the gene-editing technology CRISPR, which excels at specifically identifying genetic material.
To date, all CRISPR diagnostics also have required that the viral RNA be converted to DNA and amplified before it can be detected, adding time and complexity. In contrast, the novel approach described in this recent study skips all the conversion and amplification steps, using CRISPR to directly detect the viral RNA.
Schematic of mobile phone-based microscope for fluorescence detection showing illumination and image collection components (left). Picture of assembled device used for data collection and sample image taken by the mobile phone camera after running a Cas13a assay (right).
Dr Doudna added, "One reason we're excited about CRISPR-based diagnostics is the potential for quick, accurate results at the point of need. This is especially helpful in places with limited access to testing, or when frequent, rapid testing is needed. It could eliminate a lot of the bottlenecks we've seen with COVID-19."
Dr Parinaz Fozouni, a UCSF graduate student working in Ott's lab at Gladstone, had been working on an RNA detection 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 that testing was a potential pitfall, she and her colleagues decided to shift their focus to COVID-19.
Dr Fozouni, who is co-first author of the paper, along with Dr Sungmin Son and Dr María Díaz de León Derby from Dr Fletcher's team at UC Berkeley commented, "We knew the assay we were developing would be a logical fit to help the crisis by allowing rapid testing with minimal resources. Instead of the well-known CRISPR protein called Cas9, which recognizes and cleaves DNA, we used Cas13, which cleaves RNA."
In the novel diagnostic platform, the Cas13 protein is combined with a reporter molecule that becomes fluorescent when cut, and then mixed with a patient sample from a nasal swab. The sample is placed in a device that attaches to a smartphone. If the sample contains RNA from SARS-CoV-2, Cas13 will be activated and will cut the reporter molecule, causing the emission of a fluorescent signal. Then, the smartphone camera, essentially converted into a microscope, can detect the fluorescence and report that a swab tested positive for the virus.
Dr Ott, who is also a professor in the Department of Medicine at UCSF explained, "What really makes this test unique is that it uses a one-step reaction to directly test the viral RNA, as opposed to the two-step process in traditional PCR tests. The simpler chemistry, paired with the smartphone camera, cuts down detection time and doesn't require complex lab equipment. It also allows the test to yield quantitative measurements rather than simply a positive or negative result."
The team also says that their assay could be adapted to a variety of mobile phones, making the technology easily accessible.
Dr Fletcher further explained, "We chose to use mobile phones as the basis for our detection device since they have intuitive user interfaces and highly sensitive cameras that we can use to detect fluorescence. Mobile phones are also mass-produced and cost-effective, demonstrating that specialized lab instruments aren't necessary for this assay."
Importantly, when the researchers 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 accurately detected a set of positive samples in under 5 minutes. For samples with a low viral load, the device required up to 30 minutes to distinguish it from a negative test.
Dr Ott added, "Recent models of SARS-CoV-2 suggest that frequent testing with a fast turnaround time is what we need to overcome the current pandemic. We hope that with increased testing, we can avoid lockdowns and protect the most vulnerable populations."
Significantly, 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 become portable and eventually be made available for point-of-care or even at-home use. And, it could also be expanded to diagnose other respiratory viruses beyond SARS-CoV-2.
Furthermore 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.
Dr Ott added, "We hope to develop our test into a device that could instantly upload results into cloud-based systems while maintaining patient privacy, which would be important for contact tracing and epidemiologic studies. This type of smartphone-based diagnostic test could play a crucial role in controlling the current and future pandemics."
The team concluded, “While we demonstrate rapid detection with reasonable sensitivity using crRNAs based on existing PCR primers, we anticipate further improvement by systematically searching for the best crRNA combinations across the entire viral RNA genome. As more information becomes available about viral variants, crRNA design can be adapted to avoid false negatives. However, while combining crRNAs improves sensitivity, it also offers more opportunities for unintentional off-target detection, and lower viral loads could be registered when one crRNA in the combination does not precisely match the viral sequence in the sample. Further improvements are also anticipated in the reporter, the choice of Cas13 proteins, and in device and camera sensitivity. These advancements can improve the rate of the reaction, allowing for improvements in detection accuracy and limit of detection in shorter periods of time.”
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