A team of researchers at the Broad Institute of MIT and Harvard University have adapted the CRISPR associated RNA-cutting enzyme, Cas13, to seek out and destroy RNA-based viruses. The adapted CRISPR-Cas13 system could potentially be used as an antiviral treatment in humans.
CRISPR is best known for its use in detecting and finding rogue sections of DNA that have been inserted into the genome by viruses. The system is used in prokaryotes to repair DNA and the system has been harnessed by researchers and used to find and remove or alter mutations in the genome.
CRISPR-Cas13 has previously been adapted to detect viruses and bacteria but this is one of the first successful studies to use CRISPR-Cas13 to treat viral infections in cultured human cells.
There are currently very few FDA-approved treatments for RNA-based viruses, even though they are responsible for causing a wide range of diseases such as Ebola, Zika and influenza In the past 50 years, only 90 drugs have been developed that have been clinically approved and they are restricted to treating just 9 diseases. The FDA has also only approved vaccines to prevent infection with 16 viruses so there is a clear need for new antiviral treatment options.
RNA-based viruses can evolve quickly, so any treatment for these infections needs to also be flexible if it is to be used to treat viral infections in humans. The new system could well prove to be the flexible anti-viral treatment that is needed.
The new system has been called CARVER – Short for Cas13-Assisted Restriction of Viral Expression and Readout. CARVER can be programmed to recognize a wide range of viruses and it is quite easy to deliver into cells. While it is theoretically possible to program CARVER to perform edits on any part of a virus, since the genome changes rapidly, a section of RNA could be targeted that could ultimately have no effect.
The researchers searched for viruses that could be targeted and looked for sections of RNA that could be altered to disable the virus, while also being in parts of the virus that were least likely to mutate and render CARVER ineffective.
The researchers found hundreds of viral species that could be targeted and thousands of potential sites for edits. For the study, the researchers narrowed down those targets to three distinct types of RNA-based viruses: Influenza A virus (IAV), vesicular stomatitis virus (VSV), and lymphocytic choriomeningitis virus (LCMV).
The researchers then engineered a guide RNA for each virus, introduced their engineered Cas13 into cultured cells, then exposed the cells to the virus 24 hours later. The researchers found a 40-fold reduction in viral load 24 hours later. In a later experiment the researchers tested how much of the remaining virus was capable of infecting human cells. 8 hours following treatment, there had been a 300-fold decrease in infectivity of the influenza virus.
“Our work establishes CARVER as a powerful and rapidly programmable diagnostic and antiviral technology for a wide variety of these viruses,” said Pardis Sabeti, Broad Institute member, professor at Harvard University, and senior author of the study.
By incorporating the Cas13-based nucleic acid detection technology SHERLOCK, the researchers were able to determine the remaining levels of viral RNA in a sample.
“We envision Cas13 as a research tool to explore many aspects of viral biology in human cells,” said Catherine Freije, graduate researcher in the Sabeti lab and co-author of the study. “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.”
The study is detailed in the paper – Programmable inhibition and detection of RNA viruses using Cas13 – which was recently published in the journal Molecular Cell. DOI: 10.1016/j.molcel.2019.09.013