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Rice University, UConn Researchers Adapt CRISPR Enzyme to Create Ultra-Sensitive COVID-19 Test


NEW YORK – A research team from Rice University and the University of Connecticut has engineered a new CRISPR-based COVID-19 testing method that they say improves sensitivity and accuracy and can be applied across multiple diseases.

CRISPR as a diagnostic tool has seen a boom thanks to the COVID-19 pandemic: Multiple researchers, including teams from Stanford University and Tulane University, and companies, such as Caspr Biotech, have developed CRISPR-based SARS-CoV-2 tests. One of those assays, a high-throughput test from Mammoth Biosciences, received Emergency Use Authorization from the US Food and Drug Administration at the beginning of this year. 

Now, the Rice and UConn team has thrown their hat into the ring with a paper published last week in Nature Chemical Biology describing a method that utilizes a modified version of the CRISPR-Cas13a protein and an electrochemical sensor to detect SARS-CoV-2 RNA at very low concentrations. Xue Gao, one of the lead authors on the paper and an assistant professor of chemical and biomolecular engineering at Rice, said that the project began at the very beginning of the pandemic as she and her collaborators considered the potential applications of CRISPR for COVID-19 testing. 

Because Cas13a can work directly to find RNA targets and generate amplified and strong signals for detection, her team decided to see if they could further engineer a Cas13a protein and use it to detect RNA from the SARS-CoV-2 virus. Gao noted that the researchers didn't discover Cas13a but worked to make it more robust to "facilitate more sensitive detection." Other researchers have also used Cas13a as a tool to detect COVID-19, including a research team with collaborators from multiple universities in China and researchers from the University of California, Berkeley.

To improve the sensitivity of Cas13a, Gao and her collaborators inserted seven additional RNA binding domains into the protein to increase the ability of the protein to bind to its RNA substrates. Normally, she said, you wouldn't insert additional amino acids into big proteins like Cas13 because it could "mess up" the structure or folding of the entire protein, but the researchers were able to identify a loop region in the protein that was flexible and directly above the active site that could handle the additional binding domains. 

Those RNA binding domains act as traps that enhance the target RNA of the SARS-CoV-2 virus to bind to Cas13 while also attracting the RNA reporter molecule, which leads to a more amplified signal overall, she said. Two of the binding domains "greatly improved the detection sensitivity for different targets," including SARS-CoV-2, the researchers wrote in the Nature Chemical Biology paper.

With the additional binding domains, the "whole system is more robust and can find lower concentrations of the virus," Gao said. 

The researchers' version of Cas13a is robust, "using its own molecular machinery" and able to do its own amplification, so it doesn't require an additional RNA extraction or amplification step like PCR testing, Gao said. One of the goals was to be able to detect the RNA from a raw sample without any pretreatment — the only preparation step necessary before running the test is heating the sample for five minutes. 

The new variant of Cas13a can be used with multiple methods to detect the signal change, such as electrochemical sensors and lateral flow test strips, but the researchers chose an electrochemical sensor for the Nature Chemical Biology paper because it was "cheap and sensitive," Gao said. The fabrication cost for the entire kit was estimated at less than $5 and the turnaround time for the test was about 30 minutes. Coupling the protein with the sensor, the researchers were able to detect the virus in attomolar concentrations — more than one hundred thousandfold more sensitive than the original wild-type Cas13 protein, she noted. 

In the Nature Chemical Biology paper, the researchers applied the test to 11 confirmed positive clinical SARS-CoV-2 samples and 10 confirmed negative samples. The test detected nine of the 11 positive samples and all of the negative samples without any RNA preamplification steps — Gao said that the two positives that weren't detected had very low concentrations of the virus. The researchers have plans to further improve the ability to detect low virus concentrations, and Gao noted that a more sensitive detection device could also be used to pick up lower concentrations.

The researchers have a provisional patent for their version of the Cas13a protein with Rice and are looking to turn it into a commercial product with the university's help, Gao said. There is a commercial plan in place, with Rice helping to find diagnostic industry collaborators that could help move the test forward, she added. 

Although the current test uses an electrochemical sensor, Gao said that the protein could be further applied to a lateral flow test strip to make it more feasible for home use or low- and middle-income countries where PCR instruments and technicians are hard to find. The "final goal" is to make the test available in point-of-care settings, particularly in places where there are no easy-to-operate PCR machines.

The detection capabilities of the protein are also not limited to SARS-CoV-2, she noted. It can be applied to any nucleic acid, and researchers have already done work to use the protein to detect other viruses, such as Zika virus and influenza. Gao is also applying the technology to other diseases, including HIV and cancer detection. 

Peter Nguyen, a senior scientist at the Wyss Institute for Biologically Inspired Engineering at Harvard University, said via email that "trying to improve the collateral activity of Cas13 for diagnostics has been a really challenging problem due to the complex dynamics of the conformational changes the protein undergoes." But identifying the site where additional RNA binding domains can be inserted to improve the efficiency of the protein "potentially paves the way for further engineering of this important enzyme," he said.

Nguyen also noted that the technology can be used for COVID-19 testing and other kinds of viral or bacterial infection testing but could go beyond infectious diseases as well. "The Cas13 diagnostics can also be used to probe disease states such as cancer or other systemic diseases that may be correlated to a nucleic acid biomarker such as microRNAs," he said.

"I think where the CRISPR diagnostics shine will be point-of-care clinical testing to provide rapid nucleic acid testing with an accuracy rivaling RT-PCR without needing to send samples to a lab for processing and analysis," Nguyen added.

To get to that clinical setting, he said "the next step is to integrate the sample prep steps with their improved Cas13 enzyme and electrochemical detection into a single device." He added that it "would also be interesting to see if they can explore other RNA-binding domains to improve the activity even further."