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Genome Editing in Drug Discovery


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as well; however, the intricacies and finesses of further classification are beyond the scope of this Chapter and the reader is invited to consult recent excellent reviews on the topic (Hille et al. 2018; Koonin and Makarova 2019; Makarova et al. 2019; Nussenzweig and Marraffini 2020).

      3.3.1 crRNA Biogenesis

      In class 1 systems, the processing is performed by either Cas6 or Cas5d ribonucleases (Nam et al. 2012; Carte et al. 2008). Both of these proteins recognize and bind to the hairpin structure formed by the palindromic sequences of the pre‐cRNA, and introduce a cut immediately downstream of it (Figure 3.3a), releasing mature crRNAs (Carte et al. 2010; Haurwitz et al. 2010; Ozcan et al. 2019). Intriguingly, in CRISPR systems containing repeats which are not thermodynamically likely to form hairpin structures (namely type I‐A and ‐B, and type III‐A and ‐B) and, hence, lack inherent discriminatory borders between spacers, Cas6 seems to be able to identify repeat regions by restructuring them to favor the formation of a hairpin or hairpin‐like structure compatible with precise cleavage that will lead to productive crRNAs (Shao et al. 2016; Sefcikova et al. 2017). Mature crRNAs of most type I systems contain part of the repeat sequence at the 5’ end of the spacer and the 3’ hairpin; these do not participate in recognition of the target sequence but seem to be important for the assembly of the effector complex (Jore et al. 2011). Type III crRNA, on the other hand, undergoes additional trimming that removes the hairpin structure (Hale et al. 2008). How these mature crRNAs are paired to the cognate effector complex remains unanswered.

      Class 2 systems employ two different strategies to generate mature crRNAs. The first strategy employed by type V and VI is in principle similar to class 1 crRNA biogenesis (Figure 3.3b). Here, the effector nucleases, such as Cas12a (Cpf1) and Cas13, recognize the repeat hairpin structure within the pre‐crRNA and cleave the RNA within or upstream of it (East‐Seletsky et al. 2016; Fonfara et al. 2016).

Schematic illustration of an overview of class 1 and class 2 CRISPR systems.

      3.3.2 Interference

      3.3.2.1 Class 1

       3.3.2.1.1 Type I

Schematic illustration of crRNA biogenesis pathways.