LAUSR

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. Description CRISPR keeps on giving CRISPR RNA-guided nucleases have been the driving force behind the current revolution of genomic medicine. Hu et al. introduce a new member into the work force: a CRISPR RNA-guided protease dubbed Craspase. Combining cryo–electron microscopy and molecular genetics approaches, the study defines the conditions leading to the RNA-guided activation and inactivation of Craspase in vitro and in vivo and provides a thorough set of high-resolution mechanistic explanations for the observed activities. Because Craspases do not touch DNA, they could be a safe alternative to Cas nucleases and have the potential to be used in therapeutic applications in the future. —DJ High-resolution mechanisms are established for RNA-guided RNA and protein cleavage by CRISPR-guided Caspase (Craspase).