Scientists used two CRISPR enzymes to diagnose COVID-19 in just 20 minutes


alopah Date:2021-08-09 10:52:57 From:alopah.com
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Frequent, rapid testing for COVID-19 is reportedly essential to contain the spread of the epidemic, especially when new, more transmissible variants emerge. While today’s standard COVID-19 diagnostic test (using QRT-PCR) is very sensitive and can detect one copy of RNA per microliter, it requires specialized equipment, several hours of running time, and a centralized laboratory facility. Therefore, the test usually takes at least one to two days. A research team led by scientists from the laboratories of Jennifer Doudna, David Savage and Patrick Hsu at the University of California, Berkeley, aims to develop a diagnostic test that is faster and easier to deploy than QRT-PCR.

 

It has now combined two different types of CRISPR enzymes to create a test that can detect small amounts of viral RNA in less than an hour. Doudna won the 2020 Nobel Prize in Chemistry for developing the CRISPR-Cas9 genome editing method.

 

While the new technology has not yet reached a stage of comparable sensitivity to QRT-PCR, which can detect only a few copies of the virus per microliter of fluid, it has been able to pick up levels of viral RNA — about 30 copies per microliter — sufficient to be used to monitor populations and limit the spread of infection.

 

“You don’t need the sensitivity of PCR to basically catch and diagnose COVID-19 in the community if the test is easy and fast enough,” said David Savage, professor of molecular and cell biology and study co-author. Our hope is to push biochemistry as far as possible so that you can imagine a very convenient form where you can be tested every day, say, at the entrance to work.”

 

The researchers reported their results in The journal Nature Chemical Biology on August 5, 2021.

 

Several CRISPR-based tests have been authorized for emergency use by the U.S. Food and Drug Administration, but all require an initial step in which viral RNA is amplified so that the signal — which involves releasing a fluorescent molecule that glowed under blue light — is bright enough to be seen. While this initial amplification increased the sensitivity of the test to a level similar to that of QRT-PCR, it also introduced steps that made the test more difficult to perform outside the lab.

 

The team, led by the University of California, Berkeley, sought to achieve useful sensitivity and speed without sacrificing the simplicity of detection.

 

Team leader Tina Liu, a research scientist at the Innovative Genomics Institute (IGI) Doudna Laboratory, said: “For point-of-care applications, you want to have a quick response so that people can quickly know if they’re infected, for example, before you get on a plane or visit a relative,” said IGI, a CRISPR-focused center involving scientists from the University of California, Berkeley, and San Francisco.

 

COVID-19

 

In addition to adding an additional step, another disadvantage of initial amplification is that because it produces billions of copies of viral RNA, there is a greater chance of cross-contamination in patient samples. The new technique developed by the team reverses this and in turn enhances the fluorescent signal, eliminating a major source of cross-contamination.

 

The non-amplification technique, which they call rapid comprehensive nuclease tandem assay (find-IT), could provide a rapid and inexpensive diagnostic test for many other infectious diseases.

 

Tina said: “While we started this project with the explicit purpose of impacting COVID-19, I think this particular technology can be applied to more than just this pandemic, because ultimately, CRISPR can be programmed. So you could load the CRISPR enzyme with a sequence that targets influenza or HIV or any type of RNA virus, and the system could potentially work in the same way. This paper really establishes that this biochemistry is a much simpler way to detect RNA, and has the ability to detect that RNA in a sensitive and fast time frame, which may be suitable for future applications in point-of-care diagnostics.”

 

The researchers are currently using find-it to build such a diagnostic method, which will include the steps of collecting and processing samples and running the detection method on a compact microfluidic device.

 

To remove target amplification from the equation, the team used a CRISPR enzyme –Cas13– to detect viral RNA first, while another Cas protein called Csm6 amplified the fluorescent signal.

 

Cas13 is a general-purpose “scissors” for cutting RNA; Once it binds to the target sequence specified by the guiding RNA, it is stimulated to cut a wide range of other RNA molecules. This target-triggered cleavage activity can be used to correlate the detection of specific RNA sequences with the release of fluorescent reporter molecules. On its own, however, it may take several hours for Cas13 to produce a detectable signal when the amount of target RNA is very small.

 

Tina’s insight was to use Csm6 to amplify the effects of Cas13. Csm6 is a CRISPR enzyme that senses the presence of small circrnas and is activated to cut through a wide range of RNA molecules in cells.

 

To improve the detection of Cas13, she and her colleagues designed a specially designed activator molecule that cuts when Cas13 detects viral RNA. A fragment of this molecule binds to Csm6 and triggers Csm6 to cut and release a bright fluorescent molecule from a piece of RNA. Normally, the activator molecule is quickly broken down by Csm6, limiting the amount of fluorescent signal it can produce. Liu and her colleagues devised a way to chemically modify the activator to protect it from degradation and “charge” Csm6 to repeatedly cut and release the fluorescent molecules attached to RNA. This resulted in a sensitivity 100 times better than the original activator.

 

Tina said: “When Cas13 is activated, it cuts through this little activator, removing the section that protects it. Now that it’s liberated, it can activate a second enzyme, Csm6, many different molecules. Thus, a target identified by Cas13 not only leads to its own RNA-cutting capacity being activated; It also leads to the production of more active enzymes, each of which can then cut more fluorescent reports.”

 

The team also included an optimized guide RNA combination that made Cas13 more sensitive to the recognition of viral RNA. When this was combined with Csm6 and its activator, the team was able to detect as low as 31 copies of SARS-CoV-2 RNA per microliter in as little as 20 minutes.

 

The researchers also added RNA from patient samples to the find-it test in the microfluidic box to see if IT could be adapted to run on a portable device. Using a small device with a camera, they can detect SARS-CoV-2 RNA extracted from patient samples with a sensitivity that captures the peak of COVID-19 infection.

 

Tina stated, “This tandem nuclease method –Cas13 plus Csm6– combines everything ina single reaction at one temperature, 37 degrees Celsius, so it does not require high temperatures or multiple steps, as other diagnostic techniques do. I think this opens up the opportunity for faster, simpler tests that can achieve sensitivity comparable to other technologies today and potentially higher sensitivity in the future.”

 

The development of this amplification free RNA test resulted from a reorientation of research within IGI on the diagnosis and treatment of COVID-19 at the beginning of the pandemic. In the end, five LABS at UC Berkeley and two at UC San Francisco participated in the research project, one of many within IGI.

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