Activation of a Transcription-Dependent CRISPR-Cas System
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Date
2019-08-14
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Johns Hopkins University
Abstract
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.
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Keywords
CRISPR-Cas, CRISPR, Cas, RNA, DNA, Bacterial Immunity, Cmr, crRNA