These portable tests pair with smartphones to produce results in roughly 15 to 30 minutes. Both tests are being developed into commercial diagnostics that will be submitted to the FDA within months, the creators told IEEE Spectrum.
“CRISPR is still a relatively new method on the market, and it’s interesting to potentially be part of this first wave of CRISPR diagnostics,” says Melanie Ott, director of the Gladstone Institute of Virology and a leader of the Cell study. “Combining it with the cell phone is helping us bring diagnostics closer to people, so they are accessible for everybody.”
Here comes CRISPR
In March, biomedical engineer and diagnostics expert Tony Hu at Tulane University wondered if saliva might be able to replace nasal swabs as a COVID-19 diagnostic, since the virus infects the pulmonary system. Studies soon confirmed his suspicion that the virus was present in saliva, but the viral load in saliva ranges down to very low concentrations.
That means detecting the virus in saliva required a highly sensitive technology, Hu noted, so he turned to CRISPR, previously used to detect Zika virus. Unlike PCR, CRISPR technology can find and tag viral proteins without isolating the RNA first: If isolating RNA is like sifting through a haystack for needles, CRISPR is akin to using a magnet to simply grab them.
The Tulane test, described in the Science Advances paper, combines a saliva sample with a solution of chemicals to amplify a small region of viral RNA about a hundred million times. Next, a “guide” RNA called Cas12a finds and binds the RNA sequence for the coronavirus M protein. When a Cas12a guide grabs onto the M protein RNA, a CRISPR complex snips the piece of Cas12a and an attached DNA probe that fluoresces, like cracking a glow stick. A cell-phone camera proved to be more than sensitive enough to detect the resulting glow from the Cas12a guides, says Hu.
It sounds complicated, but it’s all done in two quick steps on a single microfluidics chip, says Bo Ning, an assistant professor at Tulane and first author on the paper. “We wanted to make the whole procedure easy to handle and friendly to use,” adds Hu.
The test produced results from a saliva sample in 15 minutes and, as hoped, detected lower concentrations of virus than traditional PCR tests. The test was also highly specific: When the team tried to trick it with over 30 other respiratory pathogens, it did not cross-react to produce a false positive result.
The assay has been licensed to NanoPin Technologies, a diagnostics company co-founded by Hu in 2017, for which he is the science advisor. Currently, NanoPin is planning to submit a lab kit version of the saliva assay for Emergency Use Authorization from the FDA by the end of January, then will move onto developing the smartphone-based version, hopefully within the next six months, says NanoPin CEO Thomas Tombler.
Detection without amplification
Two years ago, a team of scientists at Gladstone Institutes and UC San Francisco began developing an at-home test for HIV—which, like coronavirus, has RNA as its genetic material. When the pandemic hit, the team, led by Ott, bioengineer Daniel Fletcher at UC Berkeley, and CRISPR co-discoverer Jennifer Doudna at the Innovative Genomics Institute, pivoted the technology to detect SARS-CoV-2, the virus that causes COVID-19. “As soon as the genome sequence was available in January, that’s all we needed to adapt the assay to a new virus,” says Ott.
Now, they’ve done it. The assay uses three Cas13 RNA guides to hunt for bits of the virus’s RNA within a nasal swab sample. In testing, the team’s assay detected tiny amounts of virus, about 100 copies per microliter of sample, in under 30 minutes, and much larger concentrations, such as those in people who are highly contagious, in under 5 minutes.
The California test is unique in that it does not require amplifying the viral RNA first—as do PCR tests, another approved at-home test from Lucira Health, and the Tulane team’s test. By eliminating the amplification step, the results are a direct measurement of the amount of RNA in a sample, says Ott. That information can help doctors chart a patient’s course of disease and better identify appropriate treatments.
The team is now testing an in-house prototype that is intended to eventually cost less than $10 per cartridge. They expect to move forward toward FDA approval within months, says Ott. “We’re working nonstop.”
Can rapid testing save us?
Some researchers, most notably Harvard’s Michael Mina, have argued for fast-and-frequent testing using less-sensitive antigen tests for surveillance and containment. Others contend there are problems with that strategy, including limited availability of such tests and the effects of questionable accuracy.
New CRISPR-based tests like these and other molecular tests in development, if approved, could resolve those concerns and make it possible for frequent and accurate testing across the population. Both Hu and Ott imagine their tests being used at schools, airports, offices, and many other places. A cruise company has already contacted Hu about using the technology, for example. The ultimate goal for both is to develop home test kits, say Ott and Hu, but approval for those is a higher bar with the FDA, especially for a newer technology like CRISPR.