The reactions involved in the FIND-IT assay to detect infection with the SARS-CoV-2 virus. When the Cas13 enzyme (bottom left) binds to its target RNA, it snips a molecule (orange and light blue ribbon) to release an activator (orange) that supercharges the Csm6 nuclease (right) to cleave and release fluorescent molecules that light up (green) and signal the presence of viral RNA.
Frequent, rapid testing for COVID-19 is critical to controlling the spread of outbreaks, especially as new, more transmissible variants emerge.While today’s gold standard COVID-19 diagnostic test, which uses qRT-PCR—quantitative reverse-transcriptase-polymerase chain reaction (PCR)—is extremely sensitive, detecting down to one copy of RNA per microliter, it requires specialized equipment, a runtime of several hours and a centralized laboratory facility. As a result, testing typically takes at least one to two days.
While the new technique is not yet at the stage where it rivals the sensitivity of qRT-PCR, which can detect just a few copies of the virus per microliter of liquid, it is already able to pick up levels of viral RNA—about 30 copies per microliter—sufficient to be used to surveil the population and limit the spread of infections. You don’t need the sensitivity of PCR to basically catch and diagnose COVID-19 in the community, if the test’s convenient enough and fast enough. Our hope was to drive the biochemistry as far as possible to the point where you could imagine a very convenient format in a setting where you can get tested every day, say, at the entrance to work.
Several CRISPR-based assays have been authorized for emergency use by the Food and Drug Administration, but all require an initial step in which the viral RNA is amplified so that the detection signal—which involves release of a fluorescent molecule that glows under blue light—is bright enough to see. While this initial amplification increases the test’s sensitivity to a similar level as qRT-PCR, it also introduces steps that make the test more difficult to carry out outside of a laboratory.
Aside from having an added step, another disadvantage of initial amplification is that, because it makes billions of copies of viral RNA, there is a greater chance of cross-contamination across patient samples. The new technique developed by the team flips this around and instead boosts the fluorescent signal, eliminating a major source of cross-contamination.
The amplification-free technique, which they term Fast Integrated Nuclease Detection In Tandem (FIND-IT), could enable quick and inexpensive diagnostic tests for many other infectious diseases.
While we did start this project for the express purpose of impacting COVID-19, I think this particular technique could be applicable to more than just this pandemic because, ultimately, CRISPR is programable. So, you could load the CRISPR enzyme with a sequence targeting flu virus or HIV virus or any type of RNA virus, and the system has the potential to work in the same way. This paper really establishes that this biochemistry is a simpler way to detect RNA and has the capability to detect that RNA in a sensitive and fast time frame that could be amenable for future applications in point of care diagnostics.
Employing tandem Cas proteins
Cas13 is a general purpose scissors for cutting RNA; once it binds to its target sequence, specified by a guide RNA, it is primed to cut a broad range of other RNA molecules. This target-triggered cutting activity can be harnessed to couple detection of a specific RNA sequence to release of a fluorescent reporter molecule. However, on its own, Cas13 can require hours to generate a detectable signal when very low amounts of target RNA are present.
To boost Cas13 detection, she and her colleagues designed a specially engineered activator molecule that gets 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 her colleagues devised a way to chemically modify the activator so that it is protected from degradation and can supercharge Csm6 to repeatedly cut and release fluorescent molecules linked to RNA. This results in a sensitivity that is 100 times better than the original activator.
The team of researchers also incorporated an optimized combination of guide RNAs that enables more sensitive recognition of the viral RNA by Cas13. When this was combined with Csm6 and its activator, the team was able to detect down to 31 copies per microliter of SARS-CoV-2 RNA in as little as 20 minutes.
The researchers also added extracted RNA from patient samples to the FIND-IT assay in a microfluidic cartridge, to see if this assay 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 into a single reaction at a single temperature, 37 degrees Celsius, so it does not require high temperature heating or multiple steps, as is necessary for other diagnostic techniques. I think this opens up opportunities for faster, simpler tests that can reach a comparable sensitivity to other current techniques and could potentially reach even higher sensitivities in the future.
Liu, T.Y. et al. Accelerated RNA detection using tandem CRISPR nucleases. Nat Chem Biol (2021). DOI: 10.1038/s41589-021-00842-2