Researchers report that an engineered method based on CRISPR finds RNA from SARS-CoV-2, the virus that causes COVID-19, and promises to test for that and other diseases quickly and easily.
The researchers also designed the CRISPR-Cas13 RNA-modification system to enhance their power to detect minute amounts of SARS-CoV-2 virus in biological samples without the need for RNA extraction and the time-consuming amplification step of the gold standard. PCR test.
The new platform has proven to be very successful compared to PCR, finding 10 out of 11 positives and no false positives for the virus in tests on clinical samples directly from nasal swabs. The researchers demonstrated that their method detects SARS-CoV-2 markers in the atomolar (10 .).-18) concentrations.
Cas13, like its well-known cousin Cas9, is part of the system through which bacteria naturally defend themselves against phage invasion. Since its discovery, scientists have adapted CRISPR-Cas9 technology to modify living DNA genomes and show great promise in treating and even treating diseases.
It can be used in other ways. Cas13 alone can be optimized using guide RNA to find and snip the target RNA sequence, but also to find ‘collateral’, in this case the presence of viruses such as SARS-CoV-2.
“The Cas13 protein engineered in this work can be easily adapted to other previously created platforms,” says Xue Sherry Gao, associate professor of chemical and biomolecular engineering at Rice University.
“Stability and durability of engineering cup 13 The variants make it more suitable for point-of-care diagnosis in under-resourced setup areas when expensive PCR machines are not available.”
Cas13 is wild-type, taken from a bacterium, Wadi Leptotrichiaan atomolar level of viral RNA cannot be detected within a 30- to 60-minute time frame, but the new improved version does the job in about half an hour and detects SARS-CoV-2 at much lower concentrations than previous tests, says postdoctoral researcher Ji Yang.
She says the key is a flexible, well-hidden hairpin loop near the Cas13 active site. “It is in the middle of the protein near the catalytic site that determines Cas13 activity. Because Cas13 is large and dynamic, it was difficult to find a site to insert another functional domain.”
The researchers combined seven different RNA domains into the loop, and two of the complexes were clearly superior. When they find their targets, the proteins fluoresce, revealing the presence of the virus.
“We can see a five- or six-fold increase in activity compared to the wild-type Cas13,” Yang says. “This number sounds small, but it’s absolutely amazing with one step in protein engineering.
“But this was not enough for detection, so we moved the entire assay from a fluorescence plate reader, which is very large and unavailable in low-resource settings, to an electrochemical sensor, which has a higher sensitivity and can be used for point-of-care diagnostics,” she says.
With the sensor ready, Yang says the engineered protein was five times more sensitive at detecting the virus than the wild-type protein.
The lab wants to adapt its technology to paper strips like those at home COVID-19 antibody tests, but with much higher sensitivity and accuracy. “We hope that it will make this test more convenient and less costly for many purposes,” Gao says.
Researchers are also investigating improved detection of Zika, dengue and Ebola viruses and predictive biomarkers of cardiovascular disease. Their work could lead to a rapid diagnosis of the severity of COVID-19.
“Different viruses have different sequences,” Yang says. “We can design the guide RNA to target a specific sequence that we can then detect, which is the power of the CRISPR-Cas13 system.”
Since the project started as soon as the pandemic hit, SARS-CoV-2 has been a natural focus. “The technology is completely scalable for all purposes,” she says. “That makes it a very good option for detecting all kinds of different mutations or coronaviruses.”
The search appears in chemical nature biology. Additional co-authors are from the University of Connecticut and Rice.
The National Science Foundation, the Welch Foundation, and the Texas Cancer Prevention and Research Institute supported the work.
source: Rice University