Researchers from the University of Rochester Center for RNA Biology and Cornell University have found a protein that enhances the anti-viral defense of CRISPR systems, broadening our understanding of the complexities of the gene-editing tool. The team discovered that the Cas13b protein not only cuts viral RNA but communicates with another protein, Csx28, to augment its anti-viral defense.
The Csx28 protein forms a pore-like structure, and when Cas13 signals it to affect membrane permeability, it changes the membrane potential. Subsequently, this crushes metabolism and hinders energy production. A virus can’t replicate under such conditions, leading to the team’s conclusion that Csx28 enhances CRISPR-Cas13 b’s phage defense. The team’s work was published in Science.
While the researchers uncovered the structure of Csx28 using high-resolution cryo-EM, many questions remain, and the team is beginning to probe the protein’s function. The team found that when Cas13 isn’t around, Csx28 isn’t active. In fact, it remains unclear what makes Csx2 become active in defense, as well as how long it stays active and what it allows through the membrane.
Understanding the biochemistry behind the pore’s opening and closing will shed light on how CRISPR-Cas13 uses it as part of its defense and provide a jumping-off point for studying membrane proteins across other CRISPR systems.
“This finding is unexpected and raises all kinds of new questions about how bacteria protect themselves and what they are doing to survive infection,” says Mark Dumont, a professor of Biochemistry and Biophysics at URMC who has spent his career studying membrane proteins. “It is also a very interesting interface between RNA biology, CRISPR, structural biology, and membrane biology. While there is no immediate medical relevance or application, the ideas that boil up from this could be very powerful.”
The study’s corresponding author, Mitchell O’Connell, Ph.D., assistant professor of Biochemistry and Biophysics at the University of Rochester Medical Center (URMC) and a member of the UR Center for RNA Biology, explains, “This finding upends the idea that CRISPR systems mount their defense only by degrading RNA and DNA in cells and really broadens our view of how CRISPR systems may be working. When we think about CRISPR, we see Cas proteins such as Cas9 or Cas13 as the big hammer doing all the damage, but that might not be the case; we found that Cas13 and Csx28 are working together to effectively extinguish a virus.”