DNA Targeting by a Minimal CRISPR RNA-Guided Cascade

Bacteria employ surveillance complexes guided by CRISPR (clustered, regularly interspaced, short palindromic repeats) RNAs (crRNAs) to target foreign nucleic acids for destruction. Although most type I and type III CRISPR systems require four or more distinct proteins to form multi-subunit surveillance complexes, the type I-C systems use just three proteins to achieve crRNA maturation and double-stranded DNA target recognition. We show that each protein plays multiple functional and structural roles: Cas5c cleaves pre-crRNAs and recruits Cas7 to position the RNA guide for DNA binding and unwinding by Cas8c. Cryoelectron microscopy reconstructions of free and DNA-bound forms of the Cascade/I-C surveillance complex reveal conformational changes that enable R-loop formation with distinct positioning of each DNA strand. This streamlined type I-C system explains how CRISPR pathways can evolve compact structures that retain full functionality as RNA-guided DNA capture platforms. [Display omitted] •Desulfovibrio vulgaris Cas5c cleaves pre-crRNAs and stays bound to the 5′ handle•The Cas7 backbone assembles cooperatively, capped by the crRNA stem-loop•Cascade/I-C resembles type I and III complexes; large and small subunits are fused•Cryo-EM reveals R-loop stabilization by Cas8c and pronounced target DNA bending Hochstrasser et al. describe the assembly and architecture of a minimal CRISPR interference complex. Type I-C Cascade’s three subunits each play multiple functional roles. Cryo-EM structures reveal the importance of the Cas8c subunit in PAM recognition and R-loop formation.