Abstract
CRISPR-Cas systems are RNA-based immune systems that protect many prokaryotes from invasion by viruses and plasmids. Type III CRISPR systems are unique, as their targeting mechanism requires target transcription. Upon transcript binding, DNA cleavage by type III effector complexes is activated. Type III systems must differentiate between invader and native transcripts to prevent autoimmunity. Transcript origin is dictated by the sequence that flanks the 3' end of the RNA target site (called the PFS). However, how the PFS is recognized may vary among different type III systems. Here, using purified proteins and in vitro assays, we define how the type III-B effector from the hyperthermophilic bacterium Thermotoga maritima discriminates between native and invader transcripts. We show that native transcripts are recognized by base pairing at positions -2 to -5 of the PFS and by a guanine at position -1, which is not recognized by base pairing. We also show that mismatches with the RNA target are highly tolerated in this system, except for those nucleotides adjacent to the PFS. These findings define the target requirement for the type III-B system from T. maritima and provide a framework for understanding the target requirements of type III systems as a whole.
Highlights
CRISPR-Cas systems are RNA-based immune systems that protect many prokaryotes from invasion by viruses and plasmids
Activation of DNA cleavage by type III systems requires pairing of the CRISPR RNAs (crRNAs) with an RNA protospacer and is regulated by the protospacer flanking site (PFS)
We have reported previously that RNA targets lacking flanking sequences can activate the DNase function of the TmaCmr complex [25] but in other type III systems, a PFS that lacks anti-tag sequence is required for activation [26, 35, 46]
Summary
A target with anti-tag sequence at all positions but Ϫ1 to Ϫ3 triggered DNA cleavage to the same extent as a target with a fully noncomplementary PFS (Fig. 2A, target 10) These observations parallel data obtained from transformation assays in Pfu [20] and suggest that the three nucleotides at the 3Ј end of the PFS (positions Ϫ1 to Ϫ3) are important for regulating the DNase activity of the TmaCmr complex. To test if positions Ϫ2 to Ϫ5 are important for regulation of DNase activity of the TmaCmr complex, we measured the extent of DNA cleavage triggered by RNA targets with varying amounts of anti-tag sequence in these posi-. We conclude that DNA cleavage by TmaCmr is highly tolerant of mismatches with the RNA protospacer and it is likely that DNA cleavage can be activated by any invading transcript with complementarity in segment 1 and enough additional base pairs to promote complex binding
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