The research study will be released today (September 22, 2022) in the journal Nature Chemical Biology. It was led by chemical and biomolecular engineer Xue Sherry Gao at Rices George R. Brown School of Engineering and postdoctoral researchers Jie Yang of Rice and Yang Song of Connecticut.
Using structure-guided Cas13, scientists at Rice University and the University of Connecticut customized a gene editing tool to serve as an extremely delicate diagnostic test for the existence of the SARS-CoV-2 infection. They used an off-the-shelf electrochemical sensing unit to deliver results. Credit: Jie Yang/Rice University
Cas13, like its better-known cousin Cas9, is part of the system by which bacteria naturally defend themselves versus getting into phages. Since its discovery, CRISPR-Cas9 has been adjusted by scientists to edit living DNA genomes and shows fantastic promise to treat and even cure illness.
And it can be utilized in other ways. Cas13 on its own can be improved with guide RNA to discover and snip target RNA series, but likewise to find “security,” in this case the presence of viruses like SARS-CoV-2.
” The crafted Cas13 protein in this work can be readily adapted to other formerly established platforms,” Gao said. “The stability and effectiveness of crafted Cas13 variations make them more suitable for point-of-care diagnostics in low-resource setting locations when expensive PCR makers are not offered.”
Yang stated wild-type Cas13, drawn from a germs, Leptotrichia wadei, can not identify attomolar level of viral RNA within a time frame of 30 to 60 minutes, but the boosted version produced at Rice gets the job done in about half an hour and identifies SARS-CoV-2 in much lower concentrations than the previous tests.
She said the secret is a well-hidden, versatile barrette loop near Cas13s active website. “Its in the middle of the protein near the catalytic site that figures out Cas13s activity,” Yang said. “Since Cas13 is dynamic and large, it was challenging to find a website to place another practical domain.”
From left, Rice University undergraduate trainee Jeffrey Vanegas, chemical and biomolecular engineer Xue Sherry Gao and postdoctoral researcher Jie Yang led the effort to modify a gene modifying tool to work as a diagnostic test for the existence of the SARS-CoV-2 virus. Credit: Rice University
The research team merged 7 various RNA binding domains to the loop, and 2 of the complexes were plainly remarkable. When they found their targets, the proteins would fluoresce, revealing the presence of the virus.
” We might see the increased activity was 5- or six-fold over wild-type Cas13,” Yang stated. “This number seems small, but its rather astonishing with a single step of protein engineering.
” But that was still insufficient for detection, so we moved the entire assay from a fluorescence plate reader, which is quite large and not available in low-resource settings, to an electrochemical sensing unit, which has higher sensitivity and can be used for point-of-care diagnostics,” she stated.
With the off-the-shelf sensor, Yang stated the engineered protein was 5 orders of magnitude (100,000 x) more sensitive in detecting the virus compared to the wild-type protein.
The lab wishes to adjust its innovation to paper strips like those in house COVID-19 antibody tests, however with much higher sensitivity and accuracy. “We hope that will make screening easier and with lower expense for many targets,” Gao stated.
The researchers are also examining improved detection of the Zika, dengue, and Ebola viruses and predictive biomarkers for heart disease. Their work might lead to fast diagnosis of the seriousness of COVID-19.
” Different infections have various series,” Yang stated. “We can develop guide RNA to target a specific sequence that we can then discover, which is the power of the CRISPR-Cas13 system.”
Because the project started just as the COVID-19 pandemic took hold, SARS-CoV-2 was a natural focus. “The technology is quite open to all the targets,” she said. “This makes it an excellent option to identify all type of anomalies or various coronaviruses.”
” We are really thrilled about this work as a combinational effort of structure biology, protein engineering, and biomedical device development,” Gao included. “I significantly appreciate all the efforts from my laboratory members and collaborators.”
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.
Co-authors of the paper are Rice postdoctoral researcher Xiangyu Deng, undergraduate Jeffrey Vanegas, and college student Zheng You; college student Yuxuan Zhang and Zhengyan Weng of the University of Connecticut; microbiology supervisor Lori Avery and Kevin Dieckhaus, a teacher of medicine, of UConn Health; Yi Zhang, an assistant professor of biomedical engineering at the University of Connecticut; and Yang Gao, an assistant teacher of biosciences at Rice.
Xue Sherry Gao is the Ted N. Law Assistant Professor of Chemical and Biomolecular Engineering at Rice.
The National Science Foundation (2031242, 2103025), the Welch Foundation (C-1952, C-2033-20200401), and the Cancer Prevention and Research Institute of Texas (RR190046) supported the research study.
Utilizing structure-guided Cas13, researchers at Rice University and the University of Connecticut customized a gene editing tool to serve as a highly delicate diagnostic test for the existence of the SARS-CoV-2 virus. Credit: Jie Yang/Rice University
She stated the key is a well-hidden, flexible hairpin loop near Cas13s active website. “Its in the middle of the protein near the catalytic site that figures out Cas13s activity,” Yang said. “Since Cas13 is large and dynamic, it was challenging to discover a site to place another functional domain.”
Rice University postdoctoral researcher Jie Yang led an effort to adapt Cas13 genome modifying tools to work as an extremely delicate detector for the presence of the SARS-CoV-2 infection, which triggers COVID-19. Credit: Jeff Fitlow/Rice University
Cas13 Engineered To Simplify the Identification of Coronavirus
A brand-new engineered CRISPR-based approach properly discovers RNA from SARS-CoV-2, the virus that causes COVID-19. The highly delicate detector promises to make testing for COVID-19 and other diseases quickly and easy.
Partners at Rice University and the University of Connecticut further engineered the RNA-editing CRISPR-Cas13 system to improve its power for finding minute amounts of the SARS-CoV-2 infection in biological samples. A huge advantage is that it does this without the time-consuming RNA extraction and amplification action essential in gold-standard PCR screening.
The new platform was extremely effective compared to PCR screening. In fact, it discovered 10 out of 11 positives and no false positives for the infection in tests on scientific samples directly from nasal swabs. The researchers showed their method discovers indications of SARS-CoV-2 in attomolar (10-18) concentrations.