CRISPR-based Rapid COVID Diagnostics

COVID-19 testing should be done frequently and quickly to prevent outbreaks from spreading, especially as new, more transmissible variants emerge.

While the gold standard COVID-19 diagnostic test, which employs qRT-PCR (quantitative reverse-transcriptase-polymerase chain reaction), is extraordinarily sensitive, identifying one copy of RNA per microliter, it necessitates specialised equipment, a lengthy runtime, and a centralised laboratory facility. As a result, testing can take anywhere from one to two days.

A study team lead by experts from the University of California, Berkeley’s Jennifer Doudna, David Savage, and Patrick Hsu labs is working to develop a diagnostic test that is considerably faster and easier to use than qRT-PCR. It has now combined two types of CRISPR enzymes to develop an assay that can detect minuscule amounts of viral RNA in under one hour. CRISPR-Cas9 genome editing was invented by Doudna, who shared the Nobel Prize in Chemistry in 2020.

While the new technology does not yet match the sensitivity of qRT-PCR, which can detect just a few copies of the virus per microliter of liquid, it can already detect enough viral RNA — around 30 copies per microliter — to be used to monitor the population and prevent infection spread.

“If the test is accessible and rapid enough, you don’t need the sensitivity of PCR to basically catch and identify COVID-19 in the community,” said co-author David Savage, professor of molecular and cell biology. “Our goal was to push biochemistry as far as we could to the point where you could picture a really convenient format in a scenario where you could get tested every day, say, at the entrance to work,” says the author.

The findings will be published in the journal Nature Chemical Biology on August 5th.

The Food and Drug Administration has approved many CRISPR-based assays for emergency use, but they all require an initial phase in which the viral RNA is amplified enough that the detection signal — which involves the release of a fluorescent molecule that glows under blue light — is visible. While this initial amplification raises the test’s sensitivity to a level comparable to qRT-PCR, it also adds steps that make the test more difficult to perform outside of a lab.

The researchers at UC Berkeley wanted to achieve a meaningful sensitivity and speed without jeopardising the assay’s simplicity.

“You want a rapid response for point of care applications so that people can quickly know if they’re infected or not, before they get on a flight, for example, or go visit relatives,” said team leader Tina Liu, a research scientist in Doudna’s lab at the Innovative Genomics Institute (IGI), a CRISPR-focused centre involving UC Berkeley and UC San Francisco scientists.

Aside from the extra step, another downside of early amplification is the increased risk of cross-contamination between patient samples because it produces billions of copies of viral RNA. The team’s new technique reverses this and instead boosts the fluorescent signal, removing a significant source of cross-contamination.

The amplification-free technique, dubbed Fast Integrated Nuclease Detection In Tandem (FIND-IT), may enable rapid and low-cost diagnostic tests for a variety of other infectious diseases.

“While we began this project with the explicit goal of affecting COVID-19, I believe this technique is applicable to more than just this pandemic, as CRISPR is ultimately programable,” Liu explained. “So, you could load the CRISPR enzyme with a sequence specific for flu viruses, HIV viruses, or any other type of RNA virus, and the system would function similarly. This article demonstrates that this biochemistry is a more straightforward method for detecting RNA and that it is capable of detecting that RNA in a sensitive and rapid manner, making it suitable for future applications in point-of-care diagnostics.”

The researchers are currently developing such a diagnostic using FIND-IT, which will include steps for sample collection and processing, as well as running the assay on a small microfluidic device.

Utilization of tandem Cas proteins

To eliminate the need for target amplification, the team used a CRISPR enzyme — Cas13 — to detect the viral RNA and a different type of Cas protein, called Csm6, to amplify the fluorescence signal.

Cas13 is a general-purpose RNA scissors; once it binds to its target sequence, which is specified by a guide RNA, it is primed to cut a wide variety of other RNA molecules. This target-induced cutting activity can be used to link the detection of a particular RNA sequence to the release of a fluorescent reporter molecule. However, when very small amounts of target RNA are present, Cas13 can take hours to generate a detectable signal on its own.

Liu’s insight was to use Csm6 to enhance Cas13’s effect. Csm6 is a CRISPR enzyme that detects small rings of RNA and activates to cut a wide variety of RNA molecules in cells.

To increase Cas13 detection, she and her colleagues engineered an activator molecule that is cut when Cas13 detects viral RNA. A fragment of this molecule can bind to and activate Csm6, resulting in the cutting and release of a bright fluorescent molecule from a piece of RNA. Normally, Csm6 rapidly degrades the activator molecule, limiting the amount of fluorescent signal it can generate. Liu and her colleagues developed a method for chemically modifying the activator in such a way that it is protected from degradation and can be used to repeatedly cut and release fluorescent molecules associated with RNA. This results in a 100-fold increase in sensitivity over the original activator.

Liu explained that when Cas13 is activated, it cleaves this small activator, removing a segment that protects it. “Now that it has been released, it is capable of activating a large number of different molecules of the second enzyme, Csm6. Thus, when Cas13 recognises a target, it does not simply activate its own RNA-cutting ability; it generates a large number of additional active enzymes, each of which can then cleave even more fluorescent reporters.”

Additionally, the researchers used an optimised combination of guide RNAs, which enables Cas13 to recognise viral RNA with greater sensitivity. When combined with Csm6 and its activator, the team was able to detect SARS-CoV-2 RNA at levels as low as 31 copies per microliter in as little as 20 minutes.

Additionally, the researchers added extracted RNA from patient samples to the FIND-IT assay in a microfluidic cartridge to determine whether the assay could be adapted for use on a portable device. They were able to detect SARS-CoV-2 RNA extracted from patient samples with a sensitivity comparable to that of peak COVID-19 infections using a small device equipped with a camera.

“Because this tandem nuclease approach — Cas13 plus Csm6 — combines everything into a single reaction at a single temperature, 37 degrees Celsius, it eliminates the need for high temperatures or multiple steps required by other diagnostic techniques,” Liu explained. “I believe this opens the door to faster, simpler tests that are comparable in sensitivity to other current techniques and may eventually achieve even higher sensitivities.”

The development of this amplification-free method for RNA detection resulted from a refocus on COVID-19 diagnosis and treatment problems within IGI as the pandemic began. Ultimately, this research project, one of several within the IGI, involved five labs at UC Berkeley and two labs at UCSF.

“When we began this project, we hoped to develop something that was comparable to PCR but did not require amplification — that would be the dream,” said Savage, the project’s principal investigator. “And from a sensitivity standpoint, we faced a roughly ten thousandfold jump. We’ve increased it by approximately a thousandfold; we’ve decreased it by approximately three orders of magnitude. Thus, we are nearly there. That seemed nearly impossible last April, as we began to map it out.”

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