RNA-Editing Tool a Fast, Easy Test for COVID-19 and Other Diseases



The virus that causes COVID-19, SARS-CoV-2, can be precisely located using a new designed CRISPR-based technique. The extremely sensitive detector promises to make COVID-19 and other illness testing quick and simple.

Researchers from Rice University and the University of Connecticut improved the CRISPR-Cas13 RNA-editing technology to increase its capability of spotting minute quantities of the SARS-CoV-2 virus in biological samples. It does this without the time-consuming RNA extraction and amplification phase required in gold-standard PCR testing, which is a major advantage.

Comparing the novel platform to PCR testing, it was quite successful. In reality, tests on clinical samples obtained directly from nose swabs revealed 10 out of 11 positives and no false positives for the virus. The researchers demonstrated how their method can detect SARS-CoV-2 in quantities as low as attomolar (10–18).

The research will be released in the journal Nature Chemical Biology today, September 22, 2022. It was directed by postdoctoral researchers Jie Yang of Rice and Yang Song of Connecticut, as well as chemical and biomolecular engineer Xue Sherry Gao from Rice's George R. Brown School of Engineering.                               
Researchers from Rice University and the University of Connecticut adapted a gene-editing technique to function as a highly sensitive diagnostic test for the presence of the SARS-CoV-2 virus using structure-guided Cas13. To deliver findings, they used an electrochemical sensor that was readily available. Image courtesy of Jie Yang/Rice University

Cas13 is a component of the system by which bacteria naturally fight themselves against invading phages, just like its more well-known cousin Cas9. CRISPR-Cas9 has been modified by scientists to alter living DNA genomes, and it offers enormous potential for curing and treating diseases.

Additionally, it has various applications. In addition to finding and snipping target RNA sequences, guide RNA can help Cas13 discover "collateral," in this case the presence of viruses like SARS-CoV-2.

The modified Cas13 protein used in this work, according to Gao, is easily adaptable to other platforms that have already been shown successful. When expensive PCR machines are not available, tailored Cas13 versions are better suited for point-of-care diagnostics in low-resource settings due to their stability and resilience.

The enhanced version developed at Rice, according to Yang, completes the task in about 30 to 60 minutes and detects SARS-CoV-2 at much lower concentrations than the previous tests. Wild-type Cas13, derived from the bacterium Leptotrichia wadei, Yang claimed, cannot detect attomolar level of viral RNA within a time frame of 30 to 60 minutes.

She claimed that close to Cas13's active site is a cleverly concealed, flexible hairpin loop. Yang stated that the location of the catalytic site, which controls Cas13 activity, is in the middle of the protein. "It was difficult to discover a place to insert another functional domain because Cas13 is vast and dynamic."

The project to adapt a gene editing tool to function as a diagnostic test for the presence of the SARS-CoV-2 virus was led by postdoctoral researcher Jie Yang, from left, chemical and biomolecular engineer Xue Sherry Gao, and undergraduate student Jeffrey Vanegas of Rice University. Rice University as source

Seven distinct RNA binding domains were fused to the loop by the study team, and two of the complexes stood out as being clearly superior. The proteins would light when they reached their targets, indicating the presence of the virus.

In comparison to wild-type Cas13, the activity was raised five- to six-fold, according to Yang. "With just one stage of protein engineering, this number might appear little, yet it's very astounding.

However, it was still insufficient for detection, so her team switched the entire assay from a fluorescence plate reader, which is fairly bulky and not suitable for low-resource settings, to an electrochemical sensor, which is more sensitive and suitable for point-of-care diagnostics.

According to Yang, the modified protein was five orders of magnitude (100,000x) more sensitive than the wild-type protein at detecting the virus using the commercial sensor.

With significantly improved sensitivity and precision, the lab hopes to transfer its method to paper strips similar to those used in COVID-19 antibody tests performed at home. Gao stated, "We anticipate that will result in testing being more cost-effective and convenient for many targets.

Additionally, the researchers are looking into better diagnostics for the Zika, dengue, and Ebola viruses as well as cardiovascular disease prognostic biomarkers. Their findings might provide a quick assessment of the severity of COVID-19.

Different viruses have various sequences, according to Yang. "The power of the CRISPR-Cas13 system is that we can create guide RNA to target a specific sequence that we can then detect."

SARS-CoV-2 was an obvious focus, though, given the experiment got underway right as the COVID-19 pandemic started to spread. She said that the technology was suitable for all of the goals. This makes it a really good alternative for detecting various coronaviruses or alterations.

As a collaboration between structural biology, protein engineering, and the development of biomedical devices, Gao continued, "We are really excited about this study." "I sincerely appreciate the efforts of my lab mates and colleagues," the author said.

In the article "Structure-Guided Engineering of LwaCas13a with Enhanced Collateral Activity for Ultrasensitive Nucleic Acid Detection," published in Nature Chemical Biology on September 22, 2022, this is mentioned.

The study's co-authors are graduate students Yuxuan Zhang and Zhengyan Weng of the University of Connecticut, as well as microbiology director Lori Avery and professor of medicine Kevin Dieckhaus of UConn Health. They are also joined by Yi Zhang, an assistant professor of biomedical engineering at the University of Connecticut, and Yang Gao, an assistant professor of biosciences at Rice.

By RICE UNIVERSITY 

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