A new study offers important mechanistic insights into a recently discovered CRISPR-guided caspase, or Craspase. Using cryo-electron microscopy (cryo-EM), researchers explained its target RNA cleavage and protease activation mechanisms, which could lead to promising antiviral and tissue engineering tools in animals and plants.
The work was co-led by Ailong Ke, PhD, professor of molecular biology and genetics at Cornell University, and Stan J.J. Brouns, PhD, associate professor of molecular microbiology at Delft University of Technology. They published their findings in a Science article titled, “Craspase is a CRISPR RNA-guided, RNA-activated protease.”
“[W]e could use a system like this to develop many biotech and therapeutic applications if we understand all the gizmos inside this machinery,” Ke said.
The team set out to determine the cryo-EM structures of Craspase in different functional states, gathering enough “snapshots” so that they had sufficient temporal resolution to interpret its mechanisms.
“These snapshots lead to a high-definition molecular movie,” Ke said. “By watching it back and forth, we know precisely how Craspase identifies an RNA target, how this, in turn, activates the protease, how long the activity persists, and what eventually shuts the protease activity off.”
Chunyi Hu, PhD, a postdoc in Ke’s lab and one of the first authors on the paper, explained that there is tremendous interest in Craspase. “Lots of competition. We and our Netherlands collaborators pooled our strength together and worked day and night to solve the puzzle,” he said.
“A new frontier in CRISPR-Cas biology has emerged, in which the RNA-guided effectors control physiological responses using mechanisms other than nucleic acid degradation,” the researchers wrote in their paper. “Here we define how the Craspase protease is allosterically activated by target RNA recognition and inactivated by target RNA cleavage to cleave the native substrate.”
Importantly, Hu explained, “The process holds exciting potential because the output of Craspase is protein rather than DNA degradation.”
“With other CRISPR technologies, one worries whether the enzymes we use to edit our DNA are safe enough, if there might be collateral damage or off-targeting,” Ke pointed out. “With Craspase, we can achieve many of the same beneficial therapeutic outcomes without worrying about the safety of our genome.”
Ke’s team plans to delve deeper into understanding Craspase pathways and processes. But they will also move to the application side, which could include engineering in animals and plants.
“I hope more investigators will appreciate the potential of this system and join in,” Ke said. “We all think about CRISPR-guided nuclease as a tool to cure genetic diseases, but CRISPR-guided proteases could have impacts for biology in a much broader way.”