The 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 sidechain-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.

Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about 2/5 the size of Cas9 but shares similar domain organization. The associated ωRNA plays the combined role of crRNA and tracrRNA to guide dsDNA cleavage. Here we report a 2.78 Å cryo-EM structure of IscB-ωRNA bound to dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 RNPs. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9, and contacts the RNA/DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.

Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis ( Efa ) Cas1–Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. Efa Cas1–Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes Efa Cas1–Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex has a surprisingly short mean lifetime. Remarkably, transcription efficiently resolves the postsynaptic complex and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies.

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