Dutch researchers combine CRISPR and bioluminescence in an experimental test for infectious diseases

A newly developed nocturnal protein could speed up and simplify the diagnosis of viral diseases, according to researchers in the Netherlands.
Their study, published Wednesday in ACS Publications, describes a sensitive, one-step method for rapidly analyzing viral nucleic acids and their appearance using glowing bright blue or green proteins.
The identification of pathogens by detecting their nucleic acid fingerprints is a key strategy in clinical diagnostics, biomedical research, and food and environmental safety monitoring. The widely used quantitative polymerase chain reaction (PCR) tests are highly sensitive, but require sophisticated sample preparation or interpretation of results, making them impractical for some healthcare settings or resource-limited settings.
This group from the Netherlands is the result of a collaboration between scientists from universities and hospitals to develop a fast, portable and easy-to-use nucleic acid diagnostic method that can be applied in a variety of settings.
They were inspired by firefly flashes, firefly glows, and tiny stars of aquatic phytoplankton, all powered by a phenomenon called bioluminescence. This glow-in-the-dark effect is caused by a chemical reaction involving the luciferase protein. The scientists incorporated luciferase proteins into sensors that emit light to facilitate observation when they find a target. While this makes these sensors ideal for point-of-care detection, they currently lack the high sensitivity needed for clinical diagnostic tests. While the CRISPR gene editing method can provide this capability, it requires many steps and additional specialized equipment to detect the weak signal that can be present in complex, noisy samples.
Researchers have found a way to combine a CRISPR-related protein with a bioluminescent signal that can be detected with a simple digital camera. To make sure there was enough RNA or DNA sample for analysis, the researchers performed recombinase polymerase amplification (RPA), a simple technique that operates at a constant temperature of around 100°F. They developed a new platform called the Luminescent Nucleic Acid Sensor (LUNAS), in which the two CRISPR/Cas9 proteins are specific for different contiguous portions of the viral genome, each with a unique luciferase fragment attached to them above.
When the specific viral genome the investigators are examining is present, two CRISPR/Cas9 proteins bind to the target nucleic acid sequence; they become in close proximity, allowing intact luciferase protein to form and emit blue light in the presence of a chemical substrate. . To account for the substrate consumed in this process, the researchers used a control reaction that emitted green light. A tube that changes color from green to blue indicates a positive result.
The researchers tested their platform by developing the RPA-LUNAS assay, which detects SARS-CoV-2 RNA without tedious RNA isolation, and demonstrated its diagnostic performance on nasopharyngeal swab samples from COVID-19 patients. RPA-LUNAS successfully detected SARS-CoV-2 within 20 minutes in samples with an RNA viral load as low as 200 copies/μL.
The researchers believe their assay can easily and effectively detect many other viruses. “RPA-LUNAS is attractive for point-of-care infectious disease testing,” they wrote.