Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR-Cas system.

CRISPR-Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM.

The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease.

Prokaryotes are frequently exposed to potentially harmful invasive nucleic acids from phages, plasmids, and transposons. One method of defense is the CRISPR-Cas adaptive immune system. Diverse CRISPR-Cas systems form distinct ribonucleoprotein effector complexes that target and cleave invasive nucleic acids to provide immunity. The Type III-B Cmr effector complex has been found to target the RNA and DNA of the invader in the various bacterial and archaeal organisms where it has been characterized. Interestingly, the gene encoding the Csx1 protein is frequently located in close proximity to the Cmr1-6 genes in many genomes, implicating a role for Csx1 in Cmr function. However, evidence suggests that Csx1 is not a stably associated component of the Cmr effector complex, but is necessary for DNA silencing by the Cmr system in Sulfolobus islandicus. To investigate the function of the Csx1 protein, we characterized the activity of recombinant Pyrococcus furiosus Csx1 against various nucleic acid substrates. We show that Csx1 is a metal-independent, endoribonuclease that acts selectively on single-stranded RNA and cleaves specifically after adenosines. The RNA cleavage activity of Csx1 is dependent upon a conserved HEPN motif located within the C-terminal domain of the protein. This motif is also key for activity in other known ribonucleases. Collectively, the findings indicate that invader silencing by Type III-B CRISPR-Cas systems relies both on RNA and DNA nuclease activities from the Cmr effector complex as well as on the affiliated, trans-acting Csx1 endoribonuclease.