Cas13 lab engineer to simplify coronavirus identification – ScienceDaily


An engineered CRISPR-based method that finds RNA from SARS-CoV-2, the virus that causes COVID-19, promises to make testing for that and other diseases quick and easy.

Collaborators at Rice University and the University of Connecticut have designed the CRISPR-Cas13 RNA editing 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 assay.

The new platform has been 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.

The study, led by chemical and biomolecular engineer Xue Sherry Gao at the George R. Brown School of Engineering in Rice and postdoctoral researchers Ji Yang of Rice and Yang Song of Connecticut, appears in Nature Chemical Biology.

Cas13, like its well-known cousin Cas9, is part of the system through which bacteria naturally defend themselves against phage invasion. Since its discovery, CRISPR-Cas9 technology has been adapted by scientists to modify living DNA genomes and shows great promise in treating and even curing 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 platforms that have been created previously,” Gao said. “The stability and robustness of the designed Cas13 variants make them more suitable for point-of-care diagnostics in under-resourced setup areas when expensive PCR machines are not available.”

Yang said that wild-type Cas13, taken from a bacterium, Leptotrichia wadei, cannot detect an atomolar level of viral RNA within a 30- to 60-minute time frame, but the improved version created in Rice does the job in about half an hour and detects SARS. -CoV-2 at much lower concentrations than previous tests.

The switch is a flexible, well-hidden hairpin loop near the Cas13 active site, she said. “It’s in the middle of the protein near the catalytic site that determines Cas13 activity,” Yang said. “Because Cas13 is large and dynamic, it was difficult to find a site to include 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 said. “This number sounds small, but it’s absolutely amazing with one step in protein engineering.

“But this was not sufficient 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.”

With the sensor ready, Yang said 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 found in home COVID-19 antibody tests, but with much higher sensitivity and accuracy. “We hope it will make this test more convenient and cost-effective for many purposes,” Gao said.

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 said. “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.”

But because the project started as soon as the pandemic hit, SARS-CoV-2 was a natural focus. “The technology is well suited for all targets,” she said. This makes it a very good choice for detecting all kinds of mutations or different coronaviruses.

“We are very excited about this work as a joint effort of synthetic biology, protein engineering and biomedical device development,” Gao added. “I greatly appreciate all the efforts from my lab members and collaborators.”

The paper’s co-authors are Xiangyu Deng, Rice postdoctoral researcher, undergraduate Jeffrey Vanegas and graduate student Zheng You. Graduate students Yuxuan Zhang and Zhengyan Weng from the University of Connecticut; microbiology supervisor Laurie Avery and Kevin Dieckhouse, professor of medicine at UConn Health; Yi Chang, associate professor of biomedical engineering at the University of Connecticut; and Yang Gao, associate professor of biological sciences at Rice.

Xue Sherry Gao is a legal assistant professor at Ted N.

The National Science Foundation (2031242, 2103025), the Welch Foundation (C-1952, C-2033-20200401), and the Texas Cancer Prevention and Research Institute (RR190046) supported the research.



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