Craspase is a CRISPR RNA-guided, RNA-activated protease

Hu, Chunyi; van Beljouw, Sam P.  B.; Nam, Ki Hyun; Schuler, Gabriel; Ding, Fran; Cui, Yanru; Rodríguez-Molina, Alicia; Haagsma, Anna C.; Valk, Menno; Pabst, Martin; Brouns, Stan J.  J.; Ke, Ailong

doi:10.1126/science.add5064

Craspase is a CRISPR RNA-guided, RNA-activated protease

Journal Article · 16 September 2022 · Science

DOI:https://doi.org/10.1126/science.add5064· OSTI ID:1982963

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Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Department of Bionanoscience, Delft University of Technology, 2629 HZ Delft, Netherlands.; Kavli Institute of Nanoscience, 2629 HZ Delft, Netherlands.
Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk 37673, Republic of Korea.
Department of Environmental Biotechnology, Delft University of Technology, 2629 HZ Delft, Netherlands.

The CRISPR-Cas type III-E RNA-targeting effector complex gRAMP/Cas7-11 is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here, we use cryo–electron microscopy snapshots of Craspase to explain its target RNA cleavage and protease activation mechanisms. Target-guide pairing extending into the 5' region of the guide RNA displaces a gating loop in gRAMP, which triggers an extensive conformational relay that allosterically aligns the protease catalytic dyad and opens an amino acid side-chain–binding pocket. We further define Csx30 as the endogenous protein substrate that is site-specifically proteolyzed by RNA-activated Craspase. This protease activity is switched off by target RNA cleavage by gRAMP and is not activated by RNA targets containing a matching protospacer flanking sequence. We thus conclude that Craspase is a target RNA–activated protease with self-regulatory capacity.

Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States). Laboratory for BioMolecular Structure (LBMS)

Sponsoring Organization:: USDOE Office of Science (SC)

DOE Contract Number:: SC0012704

OSTI ID:: 1982963

Journal Information:: Science, Vol. 377, Issue 6612; ISSN 0036-8075

Publisher:: AAAS

Country of Publication:: United States

Language:: English

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