## 4 CRISPR/Cas Mechanism
### Expression, crRNA Generation and Interference
#### 4.2.1 Type I Systems  
In type I CRISPR/Cas systems, regions within each repeat form a stem-loop, which is bound by the processing endoribonuclease. The endoribonucleases in type I systems belong to the group of Cas6 proteins. Characterized members of this group include Cas6b in type I-B [71], Cas5d in type I-C [49], Cas6e (CasE, Cse3) in type I-E [45, 72, 73] and Cas6f (Csy4) in type I-F [42, 74]. The pre-crRNA is cleaved in a sequence-specific manner at the downstream base of the stem-loop, producing the mature crRNA consisting of a short 5' repeat handle followed by the spacer sequence and the stem-loop of the next repeat (Figure 3B) [49, 71, 74, 75]. The crRNA and the endoribonuclease stay associated, and might serve as nucleation point [48, 49, 76] for the formation of Cascade (Figure 3C). Cascade is a ribonucleoprotein complex formed by the subtype-specific Cas proteins and the crRNA, initially identified in type I-E, but later also found in type I-C and I-F [9, 45, 46, 48, 49]. The type I-E Cascade has a shape referred to as seahorse-like and has a hexameric backbone formed by Cas7 (CasC, Cse4). The stem-loop of the crRNA is bound to the endoribonuclease Cas6e, which forms the \"head\" of the seahorse. Cse1 (CasA) forms the \"tail\" and binds the 3' end of the crRNA at  
Figure 3: **crRNA generation and target interference in type I, II and III CRISPR/Cas systems**. (**a**) Transcription of the CRISPR array into a pre-crRNA; (**b**) Processing of the pre-crRNA into mature short crRNAs. In type I, RNA cleavage is performed by Cas6-homologues, which bind the repeat stem-loop and stay associated for Cascade formation. In type II, tracrRNA is required for binding and processing of the pre-crRNA by Cas9 and RNaseIII. In type III, Cas6 binds to non-structured repeats and processes the pre-crRNA into crRNA and then dissociates; (**c**) Target binding and cleavage. Type I Cascade binds the DNA target before recruiting Cas3 for degradation. In type II, Cas9 stays associated with the tracrRNA:crRNA complex after processing and subsequently binds and cleaves target DNA. The type III-B CMR-complex binds spacer sequence and targets RNA. It is hypothesized that a type III-A Csm complex forms and this system targets DNA. the other end. Cse1 is bridged to Cas6e by a Cse2 (CasB) dimer, and linked by Cas5 (CasD) to the sixth Cas7 protein of the backbone [46,47]. The I-F and I-C complexes share common features with the I-E Cascade in terms of their shape, but also show variation, which is mainly due to different proteins involved. Different numbers of subtype-specific proteins constitute Cascade, three in the type I-C, four in I-F systems, while five distinct proteins are present in the type I-E Cascade [10]. Additional variation occurs at the level of the stoichiometry of individual protein subunits in different subtype Cascade complexes. When three or four proteins are used, the endoribonuclease, with the crRNA bound via the stem-loop, is located at one end of the complex. A homo-multimer of six proteins (Csy3 and Csd2 for I-F, and I-C, respectively) forms the neck or backbone of the complex, which holds the spacer and ensures it is extended for optimal presentation to invading nucleic acids. The end of the complex is formed by a hetero-dimer [48,49]. A type I-A complex from _S. solfataricus_ (termed aCASCADE by the authors) has also been partially characterized and contained the backbone protein, Cas7 (Cas2), Cas5a, Cas5, Cas6 and crRNAs [77]. Although not directly shown, based on the data from other type I systems, the type I-A Cascade is likely to consist of the Cas6 endoribonuclease bound to the crRNA, a backbone of six Cas7 proteins and a Cas5a/Cas5 heterodimer. During the final step of interference, the type I Cascade containing a specific crRNA recognizes and binds the appropriate target DNA (Figure 3C). In type I-E systems, a short loop within CasA recognizes potential targets via the PAM sequence and it is proposed to bind them before the target DNA is incorporated into Cascade [78].