Viral antiimmunity
The new viral protein effectively suppressed the activity of CRISPR
Daria Spasskaya, N+1
American scientists have isolated a viral protein that suppresses the activity of the CRISPR-Cas13 system working with RNA. In addition, the protein allows bacteriophages to overcome the bacterial immune system and multiply, it can potentially be used to control the activity of CRISPR in human cells, suggest the authors of the article in Science (Meeske et al., A phage-encoded anti-CRISPR enables complete evasion of type VI-A CRISPR-Cas immunity).
The CRISPR-Cas system, which has recently been actively used for editing the mammalian genome, serves bacteria in nature to protect against viruses (bacteriophages). The system is an analogue of adaptive immunity, which allows you to remember former infections and, when an enemy reappears, target the Cas nuclease against foreign DNA or RNA. However, in the process of evolution, bacteriophages develop antiimmunity, which allows them to infect bacteria, despite CRISPR.
One of these evolutionary tricks was described by geneticists from Rockefeller University, led by Luciano A. Marraffini. They found in listeriophage ϕls46 (a virus that specializes in bacteria from the genus Listeria) a small protein that inhibits the nuclease of Cas13a in Listeria seeligeri cells. Similar inhibitors from the Acr protein family have already been found for Cas9, but for the first time scientists were able to isolate one for Cas13 and reveal the mechanism of its action.
The phage genome is a DNA molecule that is transcribed into RNA when it enters a cell. The protective protein Cas13 destroys phage transcripts and suppresses the reproduction of the virus. It is also interesting for its ability to recognize RNA in mammalian cells – for example, Cas13a was used to destroy RNA viruses in human cells, to turn off gene expression, as well as to detect molecules in vitro within the SHERLOCK method. Therefore, the detection of a nuclease inhibitor may be interesting not only from the point of view of evolution, but also for biotechnology.
The researchers isolated phages from a collection of listeria strains and in the next experiment isolated those that are capable of infecting Listeria seeligeri. Then the genome of the found phage ϕls46 was sequenced and genes similar to Acr from other CRISPR systems were found there. There were four such genes, and to determine which one suppresses the antiphage immunity, all four genes were individually expressed in listeria cells. The protein found in this way was named AcrVIA1, which indicates the ability to inhibit the VIA-type CRISPR system, to which the Cas13a nuclease belongs.
The most well-known CRISPR nuclease Cas9 recognizes and cuts DNA molecules, due to the presence of a short RNA seed (in human cells, its role is played by a guiding or guide RNA molecule). Nuclease Cas13a cleaves RNA molecules by a similar mechanism of action. A study of the activity and structure of the inhibitor in combination with the target showed that it suppresses the RNase activity of Cas13a due to strong binding to the place where the nuclease interacts with the seed.
In addition, the new inhibitor turned out to be very strong. Previously described Acr proteins were able to block nuclease activity only in large quantities, that is, multiple infection was required to suppress bacterial immunity. However, the inhibition of AcrVIA1 was effective enough that each phage particle could infect a bacterial cell and multiply. The authors hope that this property will also be useful for monitoring Cas13a activity in eukaryotes.
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