The ideal COVID-19 diagnostic test would be sensitive, fast, and doable at the point of care. Unfortunately, existing tests fail to check off all three boxes. A new assay, however, has been developed that comes very close. It incorporates a technology called Fast Integrated Nuclease Detection In Tandem (FIND-IT).
FIND-IT comes from the University of California (UC), Berkeley, where scientists in the laboratories of Jennifer Doudna, PhD, David Savage, PhD, and Patrick Hsu, PhD, have found a way to use two different CRISPR enzymes to detect small amounts of viral RNA—and quickly. Working together in a single assay, the enzymes—Cas13 and Csm6—can return results in less than an hour.
The assay detects viral RNA from SARS-CoV-2, the virus that causes COVID-19. But because the assay relies on CRISPR enzymes, which are programmable, it can be easily modified to detect viral RNAs associated with other diseases.
Although the assay enabled by FIND-IT is fast and convenient—and adaptable—it currently lacks the sensitivity of today’s gold standard COVID-19 diagnostic test, which relies on quantitative reverse transcriptase PCR (qRT-PCR). The qRT-PCR test can detect extremely small amounts of RNA, as little as one copy of an RNA molecule per microliter.
Nonetheless, the FIND-IT-enabled assay is nimbler than qRT-PCR. FIND-IT is a one-pot assay that does not require specialized equipment. Unlike qRT-PCR, FIND-IT doesn’t have to wait one or two days, or more, for a centralized laboratory facility to generate results.
Currently, the FIND-IT-enabled assay has difficulty detecting fewer than 30 copies of a viral RNA molecule per microliter. This level of detection, however, is sufficient to assist in viral surveillance and in limiting 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,” said Savage, professor of molecular and cell biology at UC Berkeley. “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.”
Savage is one of three co-corresponding authors of an article that appeared August 5 in Nature Chemical Biology. (The other two are Hsu and Doudna.) The article, “Accelerated RNA detection using tandem CRISPR nucleases,” reported that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation.
“Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect SARS-CoV-2 RNA extracted from respiratory swab samples with qRT-PCR-derived cycle threshold (Ct) values up to 33, using a compact detector,” the article’s authors wrote. “[Using] a single combined reaction containing both nucleases and the activator oligonucleotide … could potentially reduce costs and simplify the development of point-of-care tests compared to assays that require additional reagents for reverse transcription, target amplification, and in vitro transcription before CRISPR-based detection.”
Several CRISPR-based assays have been authorized for emergency use by the FDA, but all require an initial step in which the viral RNA is amplified so that the detection signal—which involves the 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.
“For point-of-care applications, you want to have a rapid response so that people can quickly know if they’re infected or not, before you get on a flight, for example, or go visit relatives,” said Tina Liu, the lead author of the Nature Chemical Biology paper and a research scientist in Doudna’s laboratory at the Innovative Genomics Institute (IGI).
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.
In the FIND-IT assay, the Cas13 enzyme binds to its target RNA and snips a molecule to release an activator. The activator supercharges the Csm6 nuclease to cleave and release fluorescent molecules that light up and signal the presence of viral RNA.
“We also show that this assay can be implemented in a portable device, consisting of a microfluidic chip and a compact detector, to detect viral RNA extracted from human samples,” the article’s authors noted. “This indicates that the assay is robust and simple to adapt for use in point-of-care testing workflows.”
“When we started this, we had hopes of creating something that reached parity with PCR, but didn’t require amplification—that would be the dream,” Savage remarked. “And from a sensitivity perspective, we had about a 10,000-fold gap to jump. We’ve made it about 1,000-fold; we’ve driven it down about three orders of magnitude. So, we’re almost there. Last April, when we were really starting to map it out, that seemed almost impossible.”