RNA processing and activation of Type IIIA CRISPR-Cas systems

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DNA targeting Type I and Type II CRISPR-Cas systems recognize their nucleic acid targets using complementary base pairing with their crRNAs. crRNA-DNA target pairing is initiated at the protospacer adjacent motif (PAM) from which further hybridization proceeds in a zipper-like fashion. Once the PAM distal end of the target is reached, a conformational change (locking by Type I systems, HNH-domain engagement by Type II systems) triggers cleavage factor recruitment or cleavage itself. Both, PAM recognition together with PAM-distal end verification allows hereby the target verification across its full length. Effector complexes of Type III systems bind with their crRNA to single-stranded RNA targets and rapidly degrade the bound RNA. In addition, sufficient matching between crRNA and target triggers the activation of the HD domain, an unspecific single-stranded DNAse, of the Cas10 subunit. Type III effector complexes are thought to target emerging transcripts from invader DNA and to simultaneously degrade both the transcript as well as well as the DNA. Compared to Type I and Type II systems, it is, however, rather poorly understood how targets are selected for degradation. Given the structural similarities between Type I and Type III systems we hypothesize that both systems feature similarities in their target recognition mechanism. Here, we want to resolve the mechanism that activates the HD-domain of the Type III-A effector complex (Csm) and understand how the complex maintains the activated state over long times after RNA cleavage. To this end we will employ confocal and wide-field single-molecule fluorescence experiments as well as single-molecule mechanical measurements.