October 10, 2019 -- A first-of-its-kind study conducted by researchers at the Broad Institute of MIT and Harvard demonstrates that Cas13, a class 2 CRISPR system, can be used as an antiviral in cultured human cells. The research, published in Molecular Cell on October 10, suggests that the RNA-cutting enzyme can be programmed to detect and destroy RNA-based viruses.
The researchers believed that CRISPR effectors could be repurposed to aid in defending human cells against both DNA and RNA viruses. Previous studies have shown that cas13 CRISPR possesses the ability to efficiently target and cleave RNA in several model systems, including human cells. Moreover, Cas13 orthologs have minimal off-target effects on the host transcriptome in mammalian cells. Researchers investigated the utility of Cas13 for targeting human single-stranded RNA viruses computationally, and then tested the system's ability to inhibit viral replication.
The problem with viruses
The need for new antiviral approaches is urgent. While over 90 antiviral drugs have been approved, they treat a mere nine diseases. Overall, only 16 viruses have FDA-approved vaccines. Combined with the fact that viral pathogens evolve rapidly and gain resistance easily, concerns in the scientific community are growing.
To address this concern, the researchers developed an end-to-end technology platform called Cas-13-assisted restriction of viral expression and readout (CARVER) which combines Cas13-mediated cleavage of viral RNA with rapid Cas13-based diagnostic readout using the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) platform.
"Human viral pathogens are extremely diverse and constantly adapting to their environment, even within a single species of virus, which underscores both the challenge and need for flexible antiviral platforms," says senior author Pardis Sabeti, institute member at the Broad Institute and professor at Harvard University. "Our work establishes CARVER as a powerful and rapidly programmable diagnostic and antiviral technology for a wide variety of these viruses."
Creating a three-in-one system
First, the team screened a suite of RNA-based viruses in search of viral RNA sequences that Cas13 could efficiently target. They primarily looked for pieces that are both least likely to mutate and most likely, when cut, to disable a virus. Then they computationally analyzed sequencing data to identify thousands of potential sites, in hundreds of viral species, that could be effective targets for Cas13. Next, the researchers engineered the Cas13 guide RNA for the system.
The newly programmed Cas13 was experimentally tested in human cells infected with lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). 24 hours after Cas13 was introduced into the sample, the scientists observed up to a 40-fold reduction in the level of viral RNA in culture.
Researchers performed LCMV cell culture experiments, which provided a strict test of Cas13's ability to inhibit viral RNA. The data indicated that eight hours after viral exposure, Cas13 had reduced the infectivity of the flu virus by more than 300-fold. Lastly, the team used SHERLOCK detection technology to measure viral RNA levels following Cas13 targeting in real-time. This detection system provided rapid feedback about the effectiveness of treatment and information about specific viral mutations.
"We envision Cas13 as a research tool to explore many aspects of viral biology in human cells," says Catherine Freije, a graduate student at Harvard University. "It could also potentially be a clinical tool, where these systems could be used to diagnose a sample, treat a viral infection, and measure the effectiveness of the treatment -- all with the ability to adapt CARVER quickly to deal with new or drug-resistant viruses as they emerge."
Do you have a unique perspective on your research related to Synthetic biology and CRISPR? The Science Advisory Board wants to highlight your research. Contact the editor today to learn more.