Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d

CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies. [Display omitted] •Structures of the smallest type VI CRISPR effector in guide and target-bound states•Mechanistic insights into guide RNA and target RNA recognition•Insights into apo Cas13d structural dynamics through cryo-EM and HDX-MS•Rational engineering of Cas13d for minimal coding sequence Cryo-EM structures and biochemical analysis of CRISPR-Cas13d in apo, guide-bound, and target-bound states offer insight for engineering this RNA-targeting system.