Laboratories are developing Cas13 to make it easier to identify coronavirus – ScienceDaily

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

Researchers at Rice University and the University of Connecticut have further developed the RNA-editing CRISPR-Cas13 system to increase its performance in detecting minute amounts of the SARS-CoV-2 virus in biological samples without the time-consuming RNA extraction and microbial analysis required Amplification step boost gold standard PCR tests.

The new platform was very successful compared to PCR, finding 10 out of 11 positives and no false positives for the virus in tests using clinical specimens taken directly from nasal swabs. The researchers showed that their technique detects signs of SARS-CoV-2 in the attomolar (10th-18) concentrations.

The study, which was led by chemical and biomolecular engineer Xue Sherry Gao at Rice’s George R. Brown School of Engineering and Rice postdoctoral fellows Jie Yang and Connecticut’s Yang Song, appears in Nature Chemical Biology.

Cas13, like its better-known cousin Cas9, is part of the system by which bacteria naturally defend themselves against invading phages. Since its discovery, CRISPR-Cas9 has been adapted by scientists to edit living DNA genomes and shows promise for treating and even curing disease.

And it can be used in other ways. Cas13 alone can be enriched with guide RNA to find and excise target RNA sequences, but also to find “collaterals”, in this case the presence of viruses like SARS-CoV-2.

“The engineered Cas13 protein in this work can be easily adapted to other previously established platforms,” ​​Gao said. “The stability and robustness of engineered Cas13 variants make them more suitable for point-of-care diagnostics in resource-poor areas when expensive PCR machines are not available.”

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

She said the key was a well-hidden, flexible hairpin loop near the active site of Cas13. “It’s located in the middle of the protein near the catalytic site that determines Cas13’s activity,” Yang said. “Because Cas13 is large and dynamic, finding a site to insert another functional domain was a challenge.”

The researchers fused seven different RNA-binding domains to the loop, and two of the complexes were clearly superior. When they found their targets, the proteins fluoresced, indicating the presence of the virus.

“We could see that the increased activity was five or six times that of wild-type Cas13,” Yang said. “This number may seem small, but with a single protein engineering step, it’s quite astounding.

“But that still wasn’t enough for detection, so we moved the entire assay from a fluorescence plate reader, which is quite large and unavailable in resource-poor environments, to an electrochemical sensor, which has higher sensitivity and is used for dots.” can become of-care diagnostics,” she said.

With the commercially available sensor, Yang said, the engineered protein was five orders of magnitude more sensitive in detecting the virus compared to the wild-type protein.

The lab wants to adapt its technology to paper strips like those used in home COVID-19 antibody tests, but with much greater sensitivity and accuracy. “We hope that testing will become more convenient and cost-effective for many targets,” Gao said.

Researchers are also studying improved detection of Zika, dengue and Ebola viruses and predictive biomarkers for cardiovascular disease. Your work could lead to a rapid diagnosis of the severity of COVID-19.

“Different viruses have different sequences,” Yang said. “We can design guide RNA to target a specific sequence that we can then detect, which is the power of the CRISPR-Cas13 system.”

However, since the project started just as the pandemic was taking hold, SARS-CoV-2 was a natural focus. “The technology is suitable for all targets,” she said. “That makes it a very good way to detect all kinds of mutations or different coronaviruses.”

“We are very excited about this work as a combination of structural biology, protein engineering and biomedical device development,” Gao added. “I really appreciate all the efforts of my lab members and staff.”

The work is co-authored by Rice postdoc Xiangyu Deng, student Jeffrey Vanegas, and graduate student Zheng You; University of Connecticut graduate students Yuxuan Zhang and Zhengyan Weng; Lori Avery, Chief of Microbiology, and Kevin Dieckhaus, Professor of Medicine at UConn Health; Yi Zhang, assistant professor of biomedical engineering at the University of Connecticut; and Yang Gao, assistant professor of life sciences at Rice.

Xue Sherry Gao is the Ted N. Law Assistant Professor of Chemical and Biomolecular Engineering at Rice.

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

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