Activation of a Transcription-Dependent CRISPR-Cas System
Johnson, Kaitlin N
MetadataShow full item record
CRISPR-Cas provides prokaryotes with adaptive immunity from bacteriophage and other MGEs (mobile genetic elements). CRISPR-Cas systems use a crRNA-guided effector complex to specifically target and destroy invading nucleic acids. These immune systems are immensely diverse among the six types, and often within subtypes. Whereas most types of CRISPR-Cas target foreign DNA, Type III systems (Csm/Cmr) utilize a transcriptioncoupled mechanism to target RNA. Upon target RNA binding, the Type III effector degrades both RNA and DNA and produces a secondary messenger that activates a trans-acting RNase. To prevent inappropriate activation by host-derived targets, effector activity is dependent upon identification of the target’s origin, which is signaled by the nucleotides most adjacent to the targeted sequence. Two mechanisms for this identification have been described in two subtypes of Type III systems. The complex may specifically recognize these sequences, or a host-derived sequence may base pair with the crRNA tag. It is currently unknown if the mechanism used by a given effector is subtype- or species-specific. Contrary to data available for most other CRISPR-Cas systems, it has been suggested that Type III effectors are extremely tolerant of mismatches. The data supporting this is sparse, but mismatch tolerance has been demonstrated in one subtype of Type III. It is also unknown if this is a subtype- or species-specific characteristic. Here we utilize recombinantly purified Thermotoga maritima Cmr (TmaCmr) complex and RNA targets generated by in vitro transcription to systematically explore these questions. We demonstrate that both mechanisms are utilized by TmaCmr to identify hostderived transcripts. We also show that TmaCmr is extremely tolerant of mismatches. These results unify the features of the two major subtypes of Type III CRISPR-Cas systems.