RNA activation‐independent DNA targeting of the Type III CRISPR‐Cas system by a Csm complex

The CRISPR ‐Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR ‐Cas system targets RNA and DNA in a transcription‐coupled manner, but the exact mechanism of DNA targeting by this complex remains elusive. In this study, an effector Csm holocomplex derived from Thermococcus onnurineus is reconstituted with a minimalistic combination of Csm1 1 2 1 3 3 4 1 5 1 , and shows RNA targeting and RNA ‐activated single‐stranded DNA (ss DNA ) targeting activities. Unexpectedly, in the absence of an RNA transcript, it cleaves ss DNA containing a sequence complementary to the bound cr RNA guide region in a manner dependent on the HD domain of the Csm1 subunit. This nuclease activity is blocked by a repeat tag found in the host CRISPR loci. The specific cleavage of ss DNA without a target RNA suggests a novel ss DNA targeting mechanism of the Type III system, which could facilitate the efficient and complete degradation of foreign nucleic acids.