Clustered, regularly interspaced, short palindromic repeats–CRISPR associated systems (CRISPR-Cas) are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. In fact, this system also known as RNA-targeting, where Cas9 is part of the CRISPR genetic defense system in bacteria, which scientists have been harnessing to edit DNA in animals, plants and even human cells.
The RNA-targeting technique resembles RNA interference, which scientists use as a tool for shutting off selected genes in cells, animals and plants. However, RNA interference hijacks machinery- human and animal cells use to control their own genes, and many viruses have developed sophisticated mechanisms to manipulate this machinery in their host cells.
Scientists at the University of Emory have now obtained the Cas9 enzyme from Francisella novicida, a relative of the bacterium that causes tularemia. They have adapted an antiviral enzyme from these bacteria into an instrument for inhibiting hepatitis C virus in human cells.
“We can envision using Cas9-based technology to prevent viral infections in transgenic animals and plants, for example,” says co-senior author David Weiss, PhD, assistant professor of medicine (infectious disease) at Emory Vaccine Center and Emory University School of Medicine. “This is a proof of principle that we can re-engineer Cas9 to target RNA in human or other mammalian cells. Here, we’re targeting a viral RNA, for which there is no corresponding DNA in the cells.”
In the laboratory, Cas9 and a “guide RNA” directing Cas9 against hepatitis C virus could slow down viral infection of cultured liver cells. These tools could inhibit, but not completely shut down, an established infection.
“Since Cas9 is a bacterial protein and eukaryotic viruses have likely not encountered it, they would not have ways to evade Cas9,” Weiss says. “Thus, Cas9 could be effective in inhibiting viruses when the RNAi system cannot” he added.
The list of potential uses for the CRISPR/Cas system grows longer by the day. Now fighting viruses may soon be added to that list. Last summer, another group showed that CRISPR/Cas9 could be used to seek out and excise HIV DNA from the genomes of latently – infected cells. The authors, Weiss and Grakoui, next plan to turn their efforts to targeting other RNA viruses. One goal is to express FnCas9 and appropriate guiding RNAs in crops to protect them from devastating viruses.
Based on their publication, they concluded that their data demonstrated the successful adaptation of the CRISPR-Cas prokaryotic immune system as an intracellular eukaryotic antiviral defense and suggested a portable, inter-domain machinery capable of viral inhibition; likely just one of myriad potential biotechnological and medical applications of Cas9- mediated RNA targeting. Although several effective drugs are now available to treat hepatitis C infection, the approach could have biotechnology applications.
The results were published in Proceedings of the National Academy of Sciences.