CRISPR-based test could give fast, affordable testing to help control COVID-19 spread

Overview

• Post By : Kumar Jeetendra


• Source: Gladstone Institutes


• Date: 07 Dec,2020

Imagine swabbing your nostrils, putting the swab in a device, and obtaining a read-out on your mobile phone in 15 to 30 minutes which tells you if you’re infected with the COVID-19 virus. This has been the vision for a group 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 making this vision a reality.

One of the major hurdles to fighting the COVID-19 pandemic and fully reopening communities throughout the nation is that the availability of mass rapid testing. Knowing who’s infected would provide invaluable insights regarding the possible spread and threat of this virus for policymakers and citizens alike.

Yet, individuals must often wait several days for their results, or even longer when there is a backlog in processing laboratory tests. And, the situation is worsened by the fact that the majority of infected individuals have mild or no symptoms, yet still carry and spread the virus.

In a new study published in the scientific journal Cell, the team from Gladstone, UC Berkeley, and UCSF has outlined the tech for a CRISPR-based evaluation for COVID-19 that uses a smartphone to provide accurate results in under half an hour.

The technique was created in cooperation with UC Berkeley bioengineer Daniel Fletcher, PhD, as well as Jennifer Doudna, PhD, who is a senior investigator at Gladstone, a professor at UC Berkeley, president of the Innovative Genomics Institute, and an investigator of the Howard Hughes Medical Institute.

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

“When coupled with repeated testing, measuring viral load could help determine whether a disease is increasing or decreasing,” says Fletcher, who’s also a Chan Zuckerberg Biohub Investigator. “Monitoring the course of a patient’s infection could help health care professionals estimate the stage of infection and forecast, in real time, how long is likely needed for healing.”

A simpler test through immediate detection
But 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. Additionally, this technique relies on a two-step chemical reaction, including an amplification step to provide enough of this DNA to make it detectable. So, present tests typically require trained users, technical reagents, and clumsy lab equipment, which severely limits where testing may occur and causes delays in receiving results.

All CRISPR diagnostics to date have demanded the viral RNA be converted to DNA and amplified before it can be detected, adding sophistication and time.

“One reason we are excited about CRISPR-based diagnostics is the potential for rapid, accurate results at the point of demand,” says Doudna. “This is particularly helpful in areas with limited access to testing, or when frequent, rapid testing is needed. It could eliminate a lot of the bottlenecks we have seen with COVID-19.”

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

“We knew the assay we were developing would be a logical fit to assist the crisis by enabling rapid testing with minimal resources,” states Fozouni, who’s co-first author of the paper, along with Sungmin Son and María Díaz de León Derby from Fletcher’s staff at UC Berkeley. “Rather than the well-known CRISPR protein called Cas9, which recognizes and cleaves DNA, we used Cas13, which cleaves RNA.”

If the sample contains RNA from SARS-CoV-2, Cas13 will be triggered and will reduce the reporter molecule, causing the emission of a fluorescent signal. Then, the smartphone essentially converted into a microscope, can detect the fluorescence and report that a swab tested positive for the virus.

“What really makes this evaluation unique is that it uses a one-step reaction to immediately test the viral RNA, instead of the two-step process in traditional PCR tests,” says Ott, who’s also a professor in the Department of Medicine at UCSF. “The simpler chemistry, paired with the smartphone camera, cuts detection time and doesn’t require complicated lab equipment. It also allows the test to yield quantitative measurements instead of simply a positive or negative result.”

The researchers also state that their assay could be adapted to various cellular phones, which makes the technology readily accessible.

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

Accurate and quick results to limit the pandemic
When the scientists tested their device using individual samples, they confirmed that it could offer 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 under 5 minutes. For samples with a low viral load, the apparatus took up to 30 minutes to distinguish it from a negative test.

“Recent versions of SARS-CoV-2 imply that frequent testing with a quick turnaround time is what we will need to overcome the present pandemic,” says Ott. “We hope that with increased testing, we can avoid lockdowns and protect the most vulnerable people.”

Not only does the new CRISPR-based test provide a promising choice for rapid testing, but by using a smartphone and avoiding the need for bulky laboratory equipment, it has the potential to become portable and finally be made available for point-of-care or even at-home use. And, it might also be expanded to diagnose other respiratory viruses beyond SARS-CoV-2.

In addition, the high sensitivity of smart phones, together with their own connectivity, GPS, and data-processing capacities, have made them attractive tools for diagnosing disease in low-resource regions.

“We expect to develop our test into a system that could instantly upload outcomes into cloud-based systems while maintaining patient privacy, which would be important for contact tracing and epidemiologic studies,” Ott says. “This sort of smartphone-based diagnostic evaluation could play a vital role in controlling the current and future pandemics.”