Ole Niewoehner

Prokaryotic CRISPR-Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR-Cas systems, the RNA-guided multisubunit Csm effector complex targets both single-stranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-associated protein Csm6. Here we report the crystal structure of Csm6 from Thermus thermophilus and show that the protein is a ssRNA-specific endoribonuclease. The structure reveals a dimeric architecture generated by interactions involving the N-terminal CARF and C-terminal HEPN domains. HEPN domain dimerization leads to the formation of a composite ribonuclease active site. Consistently, mutations of invariant active site residues impair catalytic activity in vitro. We further show that the ribonuclease activity of Csm6 is conserved across orthologs, suggesting that it plays an important functional role in CRISPR-Cas systems. The dimer interface of the CARF domains features a conserved electropositive pocket that may function as a ligand-binding site for allosteric control of ribonuclease activity. Altogether, our work suggests that Csm6 proteins provide an auxiliary RNA-targeting interference mechanism in type III-A CRISPR-Cas systems that operates in conjunction with the RNA- and DNA-targeting endonuclease activities of the Csm effector complex.

Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.

In many bacteria and archaea, small RNAs derived from clustered regularly interspaced short palindromic repeats (CRISPRs) associate with CRISPR-associated (Cas) proteins to target foreign DNA for destruction. In Type I and III CRISPR/Cas systems, the Cas6 family of endoribonucleases generates functional CRISPR-derived RNAs by site-specific cleavage of repeat sequences in precursor transcripts. CRISPR repeats differ widely in both sequence and structure, with varying propensity to form hairpin folds immediately preceding the cleavage site. To investigate the evolution of distinct mechanisms for the recognition of diverse CRISPR repeats by Cas6 enzymes, we determined crystal structures of two Thermus thermophilus Cas6 enzymes both alone and bound to substrate and product RNAs. These structures show how the scaffold common to all Cas6 endonucleases has evolved two binding sites with distinct modes of RNA recognition: one specific for a hairpin fold and the other for a single-stranded 5'-terminal segment preceding the hairpin. These findings explain how divergent Cas6 enzymes have emerged to mediate highly selective pre-CRISPR-derived RNA processing across diverse CRISPR systems.