Jie Yang, a Rice University postdoctoral researcher, researched the potential of modifying the Cas13 genome editing tools to serve as a highly sensitive detector for the presence of the SARS-CoV-2 virus, which causes COVID-19. Credit: Jeff Fitlow/Rice University
A new CRISPR-based technique detects COVID-19, the virus that causes it. The highly sensitive detector promises to make testing for COVID-19 and other illnesses quick and simple.
The CRISPR-Cas13 system, which is RNA-editing, has been further enhanced by Rice University and the University of Connecticut researchers in order to enhance its capability for detecting minute amounts of the SARS-CoV-2 virus in biological samples. A major advantage is that it does this without the time-consuming RNA extraction and amplification step required in gold-standard PCR testing.
Comparative to PCR testing, the new technique found ten out of eleven positives and no false positives for the virus in tests on clinical samples directly from nasal swabs. The scientists demonstrated that their technique detects signs of SARS-CoV-2 in attomolar (10-18) concentrations.
The research will be published today (September 22, 2022) in the journal Nature Chemical Biology. It was led by chemical and biomolecular engineer Xue Sherry Gao of Rice's George R. Brown School of Engineering and postdocoral researchers Jie Yang of Rice and Yang Song of Connecticut.
Researchers at Rice University and the University of Connecticut modified a gene editing tool to use as a highly sensitive diagnostic test for the presence of the SARS-CoV-2 virus. They used an off-the-shelf electrochemical sensor to obtain results.
Cas13, like its better-known cousin Cas9, is part of the bacteria's natural defense against invasion by invaded phages. Since its discovery, CRISPR-Cas9 has been modified by scientists to modify living DNA genomes, and has the potential to treat and even cure illnesses.
Cas13 is capable of being extended in other ways. For example, it may be enhanced with guide RNA to find and snip target RNA sequences, but also to discover "collateral," in this case viruses like SARS-CoV-2.
“The engineered Cas13 protein in this study is easily adaptable to other previously established platforms,” Gao said. “They are particularly suitable for point-of-care diagnostics in low-resource areas when expensive PCR machines are not available.”
Yang said that wild-type Cas13, created from a bacteria, would not be capable of detecting viral RNA within a 30 to 60 minute time frame, but an enhanced version created at Rice does the job in about half an hour and detects SARS-CoV-2 in much lower concentrations than in previous tests.
The secret to Cas13's activity is a well-hidden, flexible hairpin loop, Yang said. "It's in the middle of the protein near the catalytic site," Yang said.
Jeffrey Vanegas, a Rice University undergraduate student, chemical and biomolecular engineer Xue Sherry Gao, and postdoctoral researcher Jie Yang led the effort to modify a gene editing tool that would serve as a diagnostic test for the SARS-CoV-2 virus. Credit: Rice University
The research team fusioned seven different RNA binding domains to the loop, and two of the complexes were clearly superior. When they discovered their targets, the proteins would fluoresce, revealing the virus's existence.
Yang explained that the increase in activity was five to six times greater than in wild-type Cas13. "This number seems small, but it's quite remarkable with just one step of protein engineering.
"But that was still not enough for detection, so we switched from a fluorescence plate reader, which is quite large and not available in low-resource environments," said the researcher.
Yang claims that the engineered protein was five orders of magnitude (100,000x) more efficient in detecting the virus than the wild-type protein.
The lab wants to adapt its technology to paper strips similar to those used in home COVID-19 antibody tests, but with much greater sensitivity and accuracy. “We hope that testing will make testing easier and less costly for many purposes,” Gao said.
The researchers are also investigating the possibility of a better detection of the Zika, dengue, and Ebola viruses as well as predictive biomarkers for cardiovascular disease. Their work may aid in a quick determination 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."
SARS-CoV-2 became a natural target because the experiment began shortly after the COVID-19 epidemic. "The technology is quite adaptable to all the objectives," said the author. "This makes it a very suitable option to detect all kinds of mutations or different coronaviruses."
“We are extremely excited about this work as a result of a joint effort of structure biology, protein engineering, and biomedical device development,” Gao continued. “I greatly appreciate all of my lab colleagues and collaborators’ efforts.”
Reference: "Structure-Guided Engineering of LwaCas13a with Enhanced Collateral Activity for Ultrasensitive Nucleic Acid Detection" 22 September 2022, Nature Chemical Biology. DOI: 10.1038/s41589-022-01135-y
Rice postdoctoral researcher Xiangyu Deng, undergraduate Jeffrey Vanegas, and graduate student Zheng You are the co-authors, as well as UConn Health's microbiology supervisor Lori Avery and Kevin Dieckhaus, a professor of medicine; Yi Zhang, a graduate professor of biomedical engineering at the University of Connecticut. Yang Gao, an assistant professor of biosciences, is the authors of the paper.
Rice University's Ted N. Law Assistant Professor of Chemical and Biomolecular Engineering is Xue Sherry Gao.
The National Science Foundation (2031242, 2103025), the Welch Foundation (C-1952, C-2033, 20200401), and the Cancer Prevention and Research Institute of Texas (RR190046) all supported the study.