Rapid COVID diagnosis with CRISPR



Frequent and rapid testing for COVID-19 is essential to controlling the spread of epidemics, especially as new, more transmissible variants emerge.

While today’s benchmark COVID-19 diagnostic test, which uses qRT-PCR – the quantitative reverse transcriptase polymerase (PCR) chain reaction – is extremely sensitive, detecting up to one copy of RNA per microliter, it requires specialized equipment, a run time of several hours and a centralized laboratory. As a result, the tests usually take 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 much faster and easier to deploy than qRT-PCR. He has now combined two different types of CRISPR enzymes to create a test capable of detecting small amounts of viral RNA in less than an hour. Doudna shared the 2020 Nobel Prize in Chemistry for the invention of CRISPR-Cas9 genome editing.

Although the new technique is not yet at the stage where it rivals the sensitivity of qRT-PCR, which can only detect a few copies of the virus per microliter of fluid, it is already capable of detecting levels of RNA. viral – about 30 copies per microliter – enough to be used to monitor the population and limit the spread of infections.

“You don’t need the sensitivity of PCR to detect and diagnose COVID-19 in the community, if the test is convenient and fast enough,” said co-author David Savage, professor of molecular and cellular biology . “Our hope was to take the biochemistry as far as possible to the point where you could imagine a very practical format in a setting where you can get tested every day, say, at the entrance to work.”

The researchers will publish their results online Aug. 5 in the journal Nature Chemical Biology.

Several CRISPR-based tests have been cleared for emergency use by the Food and Drug Administration, but all require an initial step in which the viral RNA is amplified in order for the detection signal – which involves the release of a molecule. fluorescent that glows under blue light – is bright enough to see. While this initial amplification increases the sensitivity of the assay to a level similar to that of qRT-PCR, it also introduces steps that make the assay more difficult to perform outside of a lab.

The UC Berkeley-led team sought to achieve useful sensitivity and speed without sacrificing the simplicity of the test.

“For healthcare requests, you want to have a quick response so that people can quickly know whether they are infected or not, before they take a flight, for example, or go to visit relatives,” said team leader Tina Liu, a research scientist in Doudna’s lab at the Innovative Genomics Institute (IGI), a CRISPR-focused center involving scientists from UC Berkeley and UC San Francisco.

Besides having an extra step, another downside to the initial amplification is that, because it makes billions of copies of viral RNA, there is a greater chance of cross-contamination between patient samples. The new technique developed by the team reverses this and instead amplifies the fluorescent signal, thereby eliminating a major source of cross-contamination.

The no-amplification technique, which they call Fast Integrated Nuclease Detection In Tandem (FIND-IT), could allow rapid and inexpensive diagnostic tests for many other infectious diseases.

“Although we started this project for the express purpose of having an impact on COVID-19, I think this particular technique could be applicable to more than this pandemic because, at the end of the day, CRISPR is programmable,” said Liu. “So you can load the CRISPR enzyme with a sequence targeting the influenza virus or the HIV virus or any type of RNA virus, and the system has the potential to work the same way. This article really establishes that this biochemistry is a simpler way to detect RNA and has the ability to detect this RNA in a sensitive and rapid time frame that could lend itself to future applications in point of care diagnostics.

Researchers are currently building such a diagnostic using FIND-IT, which would include steps to collect and process samples and to run the assay on a compact microfluidic device.

Use of Cas proteins in tandem

To remove target amplification from the equation, the team used a CRISPR enzyme – Cas13 – to first detect viral RNA, and another type of Cas protein, called Csm6, to amplify the signal from fluorescence.

Cas13 is a general purpose scissor for cutting RNA; once it binds to its target sequence, specified by a guide RNA, it is primed to cleave a wide range of other RNA molecules. This target-triggered cutting activity can be exploited to couple the detection of a specific RNA sequence with the release of a fluorescent reporter molecule. However, on its own, Cas13 can take hours to generate a detectable signal when very low amounts of target RNA are present.

Liu’s idea was to use Csm6 to amplify the effect of Cas13. Csm6 is a CRISPR enzyme that detects the presence of small rings of RNA and activates to cleave a wide range of RNA molecules in cells.

To boost the detection of Cas13, she and her colleagues designed a specially designed activator molecule that is cut when Cas13 detects viral RNA. A fragment of this molecule can bind to and trigger Csm6 to cut and release a bright fluorescent molecule from a piece of RNA. Normally, the activator molecule is quickly broken down by Csm6, thus limiting the amount of fluorescent signal it can generate. Liu and his colleagues have devised a way to chemically modify the activator so that it is protected from degradation and can overload Csm6 to repeatedly cut and release fluorescent molecules bound to RNA. This results in a sensitivity 100 times greater than that of the original activator.

“When Cas13 is activated, it cleaves this tiny activator, removing a segment that protects it,” Liu said. “Now that it’s released, it can activate many different molecules of this second enzyme, Csm6. And thus, a target recognized by Cas13 does not only lead to the activation of its own RNA cleavage capacity; this leads to the generation of many more active enzymes which can each cleave even more fluorescent reporters.

The team of researchers also incorporated an optimized combination of guide RNAs that allows for more sensitive recognition of viral RNA by Cas13. When this was combined with Csm6 and its activator, the team were able to detect up to 31 copies per microliter of SARS-CoV-2 RNA in as little as 20 minutes.

The researchers also added RNA extracted from patient samples to the FIND-IT test in a microfluidic cartridge, to see if the test could be adapted to run on a portable device. Using a small device with a camera, they could detect SARS-CoV-2 RNA extracted from patient samples at a sensitivity that would capture COVID-19 infections at their peak.

“This tandem nuclease approach – Cas13 plus Csm6 – combines everything in a single reaction at a single temperature, 37 degrees Celsius, so that it does not require high temperature heating or multiple steps, as required. for other diagnostic techniques, “Liu said. “I think this opens up opportunities for faster, simpler tests that can achieve sensitivity comparable to other current techniques and could potentially achieve even higher sensitivities in the future.”

The development of this amplification-free method for the detection of RNA is the result of a reorientation of research within the IGI when the pandemic began towards problems of diagnosis and treatment of COVID-19. Ultimately, five UC Berkeley labs and two UCSF labs got involved in this research project, one of many within IGI.

“When we started, we were hoping to create something that would achieve parity with PCR, but didn’t require amplification – that would be the dream,” said Savage, who was the project’s principal investigator. “And from a sensitivity standpoint, we had a gap of about ten thousand times to jump. We have done this about a thousand times; we lowered it by about three orders of magnitude. So, we’re almost there. Last April, as we really started to map it, it seemed almost impossible. “

Reference: Liu TY, Knott GJ, Smock DCJ, et al. Accelerated detection of RNA using CRISPR nucleases in tandem. Nat Chem Biol. 2021: 1-7. do I: 10.1038 / s41589-021-00842-2

This article was republished from the following materials. Note: The material may have been modified for its length and content. For more information, please contact the cited source.


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