Image created by Dr. Michael J. Miller
Researchers from the Broad Institute, Harvard University, and Princeton University have developed multiple influenza tests that use CRISPR technology to detect types of flu, distinguish between flu subtypes, and identify strains that may be resistant to antiviral treatment.
The assays use the Streamlined Highlighting of Infections to Navigate Epidemics (SHINE) technology that the researchers originally developed for SARS-CoV-2 testing. Described in a paper published in the Journal of Molecular Diagnostics, the four assays were developed for the detection and differentiation of influenza A and influenza B and subtypes H1N1 (swine flu) and H3N2, and according to the paper, the assays had 100 percent concordance with quantitative RT-PCR tests.
Before the COVID-19 pandemic, the researchers had been working on applying CRISPR technology to detect influenza with partners from the US Centers for Disease Control and Prevention, but the outbreak of SARS-CoV-2 caused a change in focus, said Cameron Myhrvold, an assistant professor at Princeton and one of the test developers. Once the pandemic began to wind down, the researchers came back to their previous work on influenza and decided to apply the SHINE platform they had developed to flu testing.
Each test detects a different target: One detects influenza A, another detects influenza B, the third detects the subtype H1N1, and the fourth detects the H3N2 subtype.
To run any of the tests, a user collects a nasopharyngeal swab or saliva sample, places it into a chemical buffer solution, and treats it with heat to inactivate the virus and the nucleases present in the sample. The resulting sample is then added to a paper test strip treated with the SHINE reaction, which reverse transcribes the RNA into DNA and then transcribes it back into RNA. The Cas13 or Cas12 molecules then bind to the target and cut the reporter molecule, which produces a signal that can be interpreted. The signal can either be fluorescent, which requires an instrument to read, or visual, which can be read with the naked eye.
Myhrvold said that the researchers developed all the tests with both types of results readout to ensure performance would remain consistent regardless of the type of readout. Depending on how a user would like to utilize the test and what equipment they have available, they could choose a fluorescent or visual readout.
He noted that the fluorescent readout provides a more quantitative result, while the visual readout has no resource constraints such as an instrument or a camera required to see the results.
The test takes about 90 minutes to return a result and can be run at the point of care, including in doctors' offices and retail pharmacies, where people may exhibit signs of respiratory illness. There is some expertise required to complete the sample processing steps, such as collecting the sample and knowing how to pipette, so home or self-testing isn't yet feasible, Myhrvold said.
The researchers are attempting to further optimize the test to make it faster and easier to use for people with no training. To increase the speed, the team is investigating how to optimize the reaction conditions by changing the temperature or the enzymes used in the reaction. For ease of use, the researchers are exploring other types of readouts, such as luminescence, which is more sensitive and faster than fluorescence. However, a luminescent readout would still require some type of reader beyond the naked eye, such as a smartphone camera, he noted.
The key advantage of the tests is that they are simpler to perform than a PCR test and can provide more information than a rapid antigen test, which can only test for influenza A or B and not strain or mutation information, according to Myhrvold.
He added that right now, the researchers have no plans for commercialization or regulatory approval of the tests, although they may license the technology to another company in the future.
Although there are many existing influenza tests, since different influenza subtypes circulate each year it's "helpful to keep track of what is circulating," he noted. In addition, although resistance to the antiviral oseltamivir is rare right now, it is the frontline therapy for influenza and a test for resistance could be useful if resistant strains start to circulate widely, Myhrvold added.
Many companies and research teams are working on CRISPR-based infectious disease tests that may eventually compete with the SHINE test, and the COVID-19 pandemic accelerated that innovation. Sherlock Biosciences is a leader in the field, and the firm has licensed Myhrvold's team's technology in the past. In 2022, the firm received $2 million from the Bill and Melinda Gates Foundation to advance its instrument-free molecular diagnostics platform using CRISPR. That same year, the company licensed technology from Harvard to enable ambient temperature reactions for its nucleic acid amplification technology, which it intends to use for infectious disease diagnostics.
Sherlock also created an assay design service in 2023 that allows users to access its artificial intelligence algorithms and create their own CRISPR-based diagnostic tests.
Last year, San Diego-based molecular detection startup VedaBio raised $40 million in a Series A round to help develop and commercialize its CRISPR-based nucleic acid detection method. Meantime, Indian biotech startup CrisprBits announced last year that it had entered a strategic collaboration with MolBio Diagnostics to launch CRISPR-based point-of-care diagnostic tests for pathogen and genetic marker detection.
Myhrvold's team is also collaborating with the US Centers of Disease Control and Prevention to adapt its technology for avian and swine influenza, particularly in light of the recent avian influenza outbreak in the US. There is a "growing need to do surveillance," and the researchers are adding new tests for different types of influenza and exploring how the technology may work with different sample types, Myhrvold said.
The SHINE technology may also be adapted to other types of pathogens or diseases, although that's not a key area of focus for the researchers currently. To adapt the test to another pathogen, only the primer sequences and CRISPR RNA sequences would need to change — something Myhrvold said his team is "pretty good" at doing, since they redesigned the platform for new SARS-CoV-2 variants.
Reference
CRISPR-Based Assays for Point-of-Need Detection and Subtyping of Influenza. Yibin B. Zhang,Jon Arizti-Sanz,A'Doriann Bradley,...Tinna-Solveig F. Kosoko-Thoroddsen,Pardis C. Sabeti,Cameron Myhrvold. Journal of Molecular Diagnostics. Vol. 26 Issue 7, July 2024. p. 599-612.