Summary Recent studies on CRISPR ‐based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon S ulfolobus islandicus REY 15 A encodes three interference modules, one of type IA and two of type IIIB . Earlier we showed that type IA activity eliminated plasmid vectors carrying matching protospacers with specific CCN PAM sequences. Here we demonstrate that interference‐mediated by one type IIIB module Cmr ‐α, and a Csx 1 protein, efficiently eliminated plasmid vectors carrying matching protospacers but lacking PAM motifs. Moreover, Cmr ‐α‐mediated interference was dependent on directional transcription of the protospacer, in contrast to the transcription‐independent activities of the type IA and type IIIA DNA interference. We infer that the interference mechanism involves transcription‐dependent DNA targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S . islandicus REY 15 A which cooperate functionally by sharing a single Cas 6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers.

A novel interference mechanism by a type IIIB CRISPR‐Cmr module in Sulfolobus