1. Archaeal CRISPR-based immune systems: exchangeable functional modules
Roger A. Garrett, Gisle Vestergaard, Shiraz A. Shah 
Trends in Microbiology 
Volume 19, Issue 11, Pages (November 2011)
DOI: /j.tim
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2. Figure 1
Scheme for the three primary functions of CRISPR systems. In the adaptation step, Cas proteins excise the protospacer sequence from a foreign DNA genetic element and insert it into the repeat adjacent to the leader of the CRISPR locus. Pre-CRISPR RNAs are then transcribed from within the leader and are subsequently processed into crRNAs each carrying a single spacer sequence and part of the adjoining repeat sequence. At the interference stage, crRNAs are assembled into protein targeting complexes that anneal to, and cleave, matching spacer sequences on either invading elements or their transcripts.
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3. Figure 2
Representative gene maps of six main classes of archaeal CRISPR systems. (a) CRISPR/aCas-pCas-iCas, common in archaea; in this example those of S. islandicus are shown. (b) CRISPR/aCas-pCas-iCas-iCmr; studied experimentally in P. furiosus. (c) CRISPR/aCas-pCas-iCas-iCsm; from Caldiarchaeum subterranum. (d) CRISPR/aCas-iCsm; shown for Thermoplasma volcanium. (e) iCmr from Hyperthermus butylicus. (f) iCsm from Methanocaldococcus vulcanius. Genes encoding the functional domains are color-coded: aCas module, light blue; pCas gene, orange; iCas module, yellow; iCsm and iCmr modules, red. t.r. genes in green encode putative transcriptional regulator genes that are not considered to be part of the functional modules. R indicates proteins carrying RNA-recognition motifs (RAMPs). (a) belongs to the type I CRISPR system; (b) and (c) are mixtures of type I and type III, whereas (d–f) are classified as type III [15].
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4. Figure 3
Phylogenetic tree of the archaeal Cas10 subtypes Cmr2, Csm1 and Csx11. These are the largest and most conserved sub components of the interference modules of type III CRISPR systems, where the iCmr module has been implicated in RNA targeting [23] and the iCsm system in DNA targeting [41]. The deep branching reflects the very divergent sequences. Analysis of the five subfamilies A–E indicates strong biases in their distributions among crenarchaea and euryarchaea, and family D is archaea-specific and is present in crenarchaea, euryarchaea and unclassified archaea. The Figure is reproduced with permission from [44]. 10% indicates the amount of amino acid sequence change for the given length on the tree branches.
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5. Figure 4
Examples of genetic exchange of functional modules where amino acid sequences from shared genes in each functional module are compared [17]. (a) Comparison of the aCas and iCas modules for type I CRISPR/Cas systems of four closely related S. islandicus strains. Pairwise they show a high sequence identity of 99% for two modules, but when the two pairs are compared the combined iCas proteins remain almost identical in sequence, whereas the aCas modules show only 74% sequence similarity between the pairs, consistent with the aCas module having been exchanged for one of the group of strains [17]. (b) A similar study was performed for shared genes of four thermoneutrophilic Pyrobaculum strains, where two pairs each show similar levels of amino acid sequence similarity for their aCas and iCas modules (about 70%), but when the two pairs are compared the aCas sequences remain constant at about 70% whereas the iCas module yields only 28% similarity – indicative of the iCas modules having been exchanged. Gene contents of the two pairs of iCas modules also indicate that they belong to different subtypes. Gene modules are color-coded as in Figure 2. Abbreviations: C, CRISPR locus; t.r., transcriptional regulator (in green); n.d., gene identity not determined.
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6. Figure 5
A type III CRISPR system of the acidothermophile A. hospitalis carrying four interwoven antitoxin–toxin vapBC gene pairs that are highly divergent in sequence [52]. Functional module genes are color-coded as in Figure 2, and include genes of unknown function (grey). Numbers of repeats are indicated for each CRISPR locus.
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