The exploration & exploitation of CRISPR-Cas type III defence strategies

Abstract

Type III CRISPR-CasThe RNA targeting type III systems are thought to target MGE-derived transcripts in the context of a transcription bubble 76,77 . The seed region at the 3' region of the Cas-complex bound crRNA guide is exposed, allowing for initiation of target RNA binding before further base-pairing propagation can occur 78 . Type III effector complexes with variable backbone lengths (typically in the range of 4-6 Cas7 subunits) result in variable 3' end crRNA processing, which leads to a variable seed sequence location in crRNA guides derived from a single CRISPR spacer 78 . When compared to a fixed seed region, a variable seed implies a major hurdle for a bacteriophage to escape a certain crRNA by mutations. After base-pairing of a target RNA to the seed region, further propagation towards the Cas10-activating region (CAR) can occur. At this point, the Cas7 subunits of the type III complex generally cleave the target RNA at 6 nt intervals [79][80][81][82] . Furthermore, in the absence of 5' handle complementarity of the crRNA with the target RNA (non-self), the Cas10 subunit of the type III complex is activated. This Cas10 subunit typically contains a HD domain, activation of which results in cleavage the exposed ssDNA regions in the transcription bubble in a sequence non-specific manner. On top of this, the activation of the Palm domain leads to cyclase activity whereby ATP is used to generate cyclic oligoadenylate (cOA) messenger molecules, which can consist in different sizes ranging from two to six adenosine moieties 73,83 . Subsequently, cOAs bind and allosterically activate proteins containing CARF [CRISPR-Associated Rossmann Fold] or SAVED [SMODS-(Second Messenger Oligonucleotide or Dinucleotide Synthetase) Associated and fused to Various Effector Domains] sensory domains. These sensory domains exist as fusions to a wide range of catalytic domains 75,84 . Recently, a novel signalling molecule (SAM-AMP) was found to be produced by a Cas10 of type III complex, including a compatible allosterically activatable ancillary effector protein 85 . A commonality is that the downstream effector modules are prolific in their catalytic activity, so much so that the cellular processes are disrupted to the point of bringing the cells to a state of dormancy or even cell death [86][87][88][89][90][91][92][93] . Ultimately, this type of abortive infection mechanism sacrifices the individual cell to protect the rest of the population. S A Chapter 2Coevolution between bacterial CRISPR-Cas systems and their bacteriophages