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Synthesis of crRNAs starts with the transcription of the CRISPR array from a promoter usually located within the leader sequence (Pul et al. 2010; Pougach et al. 2010). Processing of the pre‐crRNA is specific to each of the CRISPR class, with class 1 pre‐crRNAs cleaved by dedicated endoribonuclease, whereas the class 2 systems employ the same machinery that performs target destruction (Figure 3.3).

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).

A more elaborate strategy is used to generate mature crRNAs in type II and type V‐B systems (Figure 3.3c). These two systems, exemplified by Cas9 and Cas12b (C2c1), require a second noncoding trans‐activating CRISPR RNA (tracrRNA) to pair with the repeat regions within the pre‐crRNA and form an intermediary between the crRNA and the effector protein (Deltcheva et al. 2011; Shmakov et al. 2015). The stem‐loops of the tracrRNA act as a recruitment site for Cas9 and Cas12b, permitting them to form a ternary pre‐crRNA:tracrRNA:Cas effector complex. The binding of the effector protein further stabilizes the interaction between pre‐crRNA and tracrRNA, but also recruits cellular RNase III that cleaves the RNA:RNA duplex formed by the repeat sequences of the pre‐crRNA and tracrRNA, releasing the 3’ end of the crRNA (Deltcheva et al. 2011). The 5’ end of the crRNA is processed further by removing the remaining repeat sequence, but the protein involved has remained elusive (Hille et al. 2018; Nussenzweig and Marraffini 2020). Once fully processed, crRNA paired with its cognate effector protein can patrol the cytosol and confer immunity to any invading DNA.

Figure 3.2 Overview of class 1 and class 2 CRISPR systems. General composition of various CRISPR systems across two classes and six types. The top panel shows a legend with a simplified, hypothetical CRISPR system containing key functional modules used in the immune response (adaptation, expression, interference, or ancillary). Functions of homologous genes are distinguished by color and demarcated by shaded areas. Many Cas proteins perform multiple activities in the immune response, and are as such highlighted as multicolored fusions spanning several categories. Many components of CRISPR systems are absent from specific subtypes, and are therefore represented in washout and with a dashed outline and an asterisk. Depicted CRISPR loci are schematic and do not correspond to real‐life examples; hence the gene order, size, and orientation are purely didactic. Nomenclature and general module organization are from the most recent classification (Makarova et al., 2019).