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The most recently fully characterized CRISPR systems belong to type VI (Meeske et al. 2019). Similar to other class 2 proteins, the type VI effector Cas13 (with representative Cas13a and Cas13b) also forms a bilobed structure with the crRNA accommodated in its central cleft (Liu et al. 2017b; Liu et al. 2017c; Slaymaker et al. 2019). Unlike Cas9 and Cas12 effectors, Cas13 lacks DNase domains such as HNH or RuvC, but contains two HEPN domains, often found in RNases (Shmakov et al. 2017; Makarova et al. 2019), allowing this enzyme to target ssRNA (Abudayyeh et al. 2016; East‐Seletsky et al. 2016; Smargon et al. 2017; Zhang et al. 2018). Additionally, Cas13 proteins contain an alpha‐helical domain intuitively called Helical‐1 domain, which together with HEPN‐2 domain participates in pre‐crRNA processing. Binding of pre‐crRNA in the central channel of Cas13 leads to a conformational change that activates the Helical‐1 domain, stimulating it to incise the pre‐crRNA generating a mature crRNA (typically with 28–30 nt spacer and 30 nt long hairpin) (East‐Seletsky et al. 2016). The structure of the crRNA differs between two prominent subcategories, with the hairpin‐forming repeat sequence being at the 5’ or 3’ end of the spacer in Cas13a or Cas13b processed RNA, respectively (East‐Seletsky et al. 2016; Slaymaker et al. 2019).

Recognition of the target RNA bears similarity to other CRISPR systems (Figure 3.5c). The pairing between the crRNA and target RNA at the central seed sequence is essential for binding and then stimulating RNase activity (Abudayyeh et al. 2016; Tambe et al. 2018), whereas peripheral mismatches are tolerated to a greater extent. Further elements are also needed for activation of RNase activity of the HEPN domains. Sequence and structure of the hairpin are critical, where reducing the length under 24 nt or mutating key nucleotides significantly decreased its activity (Abudayyeh et al. 2016; Smargon et al. 2017). Analogous to PAM, Cas13 also requires protospacer flanking site (PFS) proximal to the target site; the exact sequence and position is dependent on the subtype and species. For example, Cas13a requires a 3’ non‐G PFS, while Cas13b need a PFS at either side of the protospacer, with the 5’ being depleted of C and the 3’ PFS having a consensus sequence of NAN or NNA, where N is any nucleotide (Abudayyeh et al. 2016, Smargon et al. 2017).

Productive base pairing between crRNA and target RNA triggers a conformational change bringing both of the HEPN domains in close proximity and assembling into a composite catalytic site on the outer surface of the protein (Liu et al. 2017b; Liu et al. 2017c). Activated HEPN domains can indiscriminately degrade ssRNA, both the target at single‐stranded regions and any other RNA in the vicinity (Abudayyeh et al. 2016; East‐Seletsky et al. 2016). This indiscriminate RNase activity was shown to be able to restrict invasion of RNA phages, such as MS2 (Abudayyeh et al. 2016). Furthermore, a recent study showed that type VI systems can confer immunity to DNA phages as well. Similarly to type III systems, indiscriminate RNase activity of Cas13 is able to induce growth arrest of the infected cells. While the growth arrest induced by type III systems is transient as Csm6 (which also contains HEPN domains) gets deactivated, Cas13 does not (likely due to persistent transcription from the phage genome, which remains intact), consequentially inducing dormancy. This way, the further expansion of the phage is prevented, achieving immunity on a population level (Meeske et al. 2019).