Scientists have developed a method for combating single-stranded RNA viruses using the CRISPR / Cas13 system. They found that the system successfully attacks conservative sequences in the viral genome, regardless of its size and organization, and used it against the lymphocytic choriomeningitis viruses, vesicular stomatitis and type A influenza virus. The amount of viral RNA in cell cultures fell tenfold.
The CRISPR / Cas9 system has repeatedly proven itself in editing DNA from various organisms. But in addition to correcting mutations in the body’s own genes, it can also be used to combat alien, viral genes. However, about two-thirds of all viruses attacking humans consist of a single RNA strand that multiplies on its own, without a DNA messenger. Against such an adversary, CRISPR / Cas9 is powerless.
Catherine Freije (Catherine Freije) with colleagues from the Massachusetts Institute of Technology and Harvard decided to use another bacterial enzyme – Cas13, which is able to introduce breaks in the RNA strand. Scientists called their system CARVER (Cas13-assisted restriction of viral expression and readout, “Limiting the reproduction and transmission of information from viruses using Cas13”).
To begin with, they scanned the genomes of more than 350 RNA viruses in search of sequences that would be “convenient” to sit on the Cas13 enzyme. In the genome of 95 percent of viruses, they found at least 10 of these sites. To test the method, the researchers took three viruses with different genome structures and life cycles: type A influenza viruses, lymphocytic choriomeningitis, and vesicular stomatitis. For each of them, scientists created a set of CRISPR-RNAs, which, like the guiding RNAs in the CRISPR / Cas9 system, were supposed to point “molecular scissors” to the target for destruction.
The cycle of the choriomeningitis virus completely passes through the cytoplasm; its RNA does not enter the cell nucleus. 5 out of 6 test CRISPR-RNAs together with Cas13 reduced the amount of viral RNA in infected cells by 2-14 times.
Type A influenza virus multiplies in the nucleus, and information is read from its RNA in the cytoplasm. Nevertheless, the method was again successful: scientists were able to reduce its concentration by 7-22 times. In the case of the vesicular stomatitis virus, the amount of viral RNA decreased by 7-43 times.
Researchers compared the effect of different CRISPR-RNAs and found that those that target conserved sequences in the genome of viruses, that is, those that are common to different virus lines, work best. Scientists have suggested that a CRISPR / Cas13 attack could accelerate their evolution. They isolated viral RNA from cells in which molecular scissors worked, but did not detect accumulation of mutations in these conserved regions.
The authors of the work believe that their method works efficiently and does not cause resistance in target viruses. And despite the fact that in vivo genetic editing technologies are still only being tested, researchers believe that once their method can, if not replace, then work along with vaccination, especially in cases where antiviral drugs do not exist, as is often the case with RNA -containing single-chain viruses.
Previously, scientists have already used the CRISPR / Cas9 system in order to cleanse the genome of other viruses: for example, human cells saved herpes viruses, and mice got rid of HIV. There are other projects, for example, to remove viruses from the genome of pigs in order to use their organs for xenograft.
Via | Journal – Science Direct