Preventing Autoimmunity in Transcription‐Dependent CRISPR‐Cas Systems

Abstract

The CRISPR‐Cas systems are a collection of RNA‐based adaptive immune systems that protect many prokaryotes from invasion by viruses and plasmids. CRISPR‐Cas systems are very diverse and can be organized into six types and more than 15 subtypes. Type III systems are unique as their targeting mechanism requires transcription of the target DNA. Type III effector complexes use small CRISPR RNAs (crRNAs) to identify complementary transcripts. Transcript binding simultaneously activates two nuclease domains, leading to the degradation of both the bound transcript and adjacent DNA. Like all other immune systems, CRISPR‐Cas systems must differentiate between self and non‐self in order to prevent autoimmunity, which could arise from targeting of the host CRISPR‐array. Type III systems use two mechanisms to identify self‐transcripts. Studies of the Type III‐A system from Staphylococcus epidermidis revealed that base pairing between the crRNA and the sequence flanking the 3′ end of RNA target site prevents autoimmunity. However, a study of the Type III‐B system from Pyrococcus furiosus revealed that a short sequence motif flanking the RNA target sequence is required for immunity and the absence of this motif in CRISPR arrays prevents autoimmunity. Neither of these mechanisms is well understood biochemically and it is not clear if these mechanisms are conserved within the subtypes. To address these points, we monitored RNA‐activated DNA cleavage by the Type III‐B effector complex from Thermotoga maritima with purified components. A series of RNA transcripts containing various target 3′‐flanking sequences were tested for their ability to activate DNA cleavage. We find that two factors are important in preventing autoimmunity in the T. maritima Type III‐B system: (1) base pairing between the crRNA and the target 3′‐flanking sequence of the transcript and (2) the presence of a G, which does not base pair with the crRNA, immediately adjacent to the 3′‐end of the RNA target sequence. These data suggest that the mechanisms used to avoid autoimmunity by Type III systems are not subtype‐specific and that identification of self transcripts is complex, involving base pairing and specific sequence recognition.Support or Funding InformationThis work was supported by National Institutes of Health grant GM097330 to S.B.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.