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crRNA (tracrRNA), a noncoding RNA that coordinates the processing of crRNA and whose hairpin Cas9 binds to (Deltcheva et al. 2011). Once bound to crRNA:tracrRNA complex, Cas9 identifies target DNA through PAM recognition, and base pairing between the crRNA and the target DNA (Figure 3.5a). If sufficient complementarity is present, S. pyogenes Cas9 generates a blunt DNA double‐stranded break (DSB) 3 bp upstream of the PAM through concerted activity of its RuvC and HNH domains (Jinek et al. 2012). Similarly to other systems, cleavage by Cas9 initiates further degradation by the host machinery, neutralizing the invading genome (Figure 3.5a).

      Detailed structural and biochemical studies of SpyCas9 protein have revealed a bilobed structure with the crRNA and target DNA accommodated in the central cleft. Cas9:crRNA:tracrRNA complex recognizes the PAM site via interaction of the GG dinucleotides and the conserved amino‐acids of the C‐terminal domain. Recognition of the correct PAM leads to a local unwinding of the DNA duplex, allowing the crRNA to pair with a 10–12 nt long seed sequence of the target strand (Anders et al. 2014; Sternberg et al. 2014). Successful pairing accompanied by further conformational changes prompts further invasion of the crRNA, forming a stable R‐loop across the full length of crRNA (Jiang et al. 2016a; Mekler et al. 2017). This in turn induces another set of complex conformational changes, where the movement of the HNH domain leads to activation of the RuvC domain, allowing coordinated cleavage of the target and nontarget strand, respectively (Sternberg et al. 2015; Raper et al. 2018). Biochemical studies have shown that once bound to the target DNA, cleavage is performed rapidly, while the enzyme remains stably bound, indicative of single‐turnover enzyme kinetics (Jinek et al. 2012; Raper et al. 2018). Of note, single‐turnover kinetics of Cas9 proteins is not a universal property, as an orthogonal Cas9 from S. aureus (SauCas9) shows a multiple‐turnover kinetics (Yourik et al. 2019). Furthermore, it seems that in vivo the bacterial transcriptional machinery can remove bound Cas9, making the enzyme multi‐turnover, thus enhancing the immune response (Clarke et al. 2018). Recently, the eukaryotic histone chaperone complex FACT, commonly associated with active transcription (Belotserkovskaya et al. 2003), has been shown to evict Cas9 bound to DNA, improving the enzymes’ kinetic and changing the editing outcome (Wang et al. 2020).

      3.3.2.2.2 Type V

      Type V CRISPR systems contain some of the most diverse interference modules, with a total of 17 subtypes characterized so far (Burstein et al. 2017; Harrington et al. 2018; Makarova et al. 2019; Yan et al. 2019). The effector proteins (named Cas12a‐k) differ fundamentally from type II systems by the domain architecture; while type II systems perform cleavage of target DNA by two different domains, Cas12 proteins have a single RuvC‐like domain which is able to cleave both strands (Swarts et al. 2017). The best understood (both structurally and biochemically) are Cas12a (previously known as Cpf1) and Cas12b (C2c1) proteins. Both proteins share a similar bilobed structure with Cas9, however with only one lobe containing the catalytic RuvC domain (Liu et al. 2017a).

Cas12a effector complex has been proposed to associate unspecifically to DNA and diffuse along it until a PAM sequence is found. This initiates a local unwinding and base pairing between the crRNA and target DNA (Figure 3.5b). Cas12a performs multiple checks for target recognition during the pairing of the crRNA with the 3–5 nt long seed sequence proximal to the PAM, and then during the formation of the R‐loop (Stella et al. 2018). Failure to form at least 17 bp long DNA:RNA hybrid will lead to rapid dissociation of the Cas12a:crRNA complex off DNA (Jeon et al. 2018, Singh et al. 2018). If a sufficiently long R‐loop is formed, Cas12a is activated and proceeds to cleave the nontarget strand, and then the target strand (Jeon et al. 2018), generating a staggered DNA cut with 4–5 nt long 5’ overhangs between the 18^th and 23^rd base from the PAM (Zetsche et al. 2015). After successful cleavage, Cas12a releases the PAM‐distal DNA fragment but continues to bind to the PAM‐proximal fragment (Figure 3.5b). Crucially, crRNA remains bound to the target strand, allowing the enzyme to remain in catalytically active state. This allows Cas12a proteins to exhibit an indiscriminate nuclease activity on any ssDNA it may encounter (Chen et al. 2018). This activity might be important for the complete clearance of invading genomes. Importantly, the indiscriminate ssDNase activity has been reported for many Cas12 proteins: Cas12b (Li et al. 2019a), Cas12f (Harrington et al. 2018), and Cas12c, ‐h, ‐i, and g (Yan et al. 2019). Furthermore, Cas12g was shown recently to be able to target RNA molecules, and to pose indiscriminate cleavage of ssDNA and ssRNA. Many type II proteins can target both ssDNA and dsDNA (Ma et al. 2015).

Figure 3.5 Interference mechanisms in class 2 systems. Representative mechanisms of the most commonly used class 2 systems are depicted here. In (a), Cas9:crRNA:tracrRNA binds to the target sequence, activating the RuvC and HNH nuclease domain to generate a blunt double‐stranded DNA break proximal to PAM site. In (b), Cas12a complexed with its cognate crRNA recognized target sequence, inducing cleavage of nontarget strand by its single RuvC domain. The enzyme then rearranges to cleave the target strand, releasing the PAM‐distal DNA fragment. The enzyme remains active as it still binds activating PAM‐proximal sequence, exhibiting collateral activity on any ssDNA able to enter the active site. (c) depicts a general mechanism of Cas13 enzymes, able to target RNA by base pairing with its crRNA. Upon recognition of target RNA, Cas13 enzyme undergoes a major conformational change that assembles a composite HEPN domain, exhibiting indiscriminate ssRNA nucleolytic cleavage. Activated Cas13 is therefore able to degrade the target RNA molecule, but also any bystander RNA. Sites of DNA cleavage activity are depicted by orange arrows and RNA cleavage by red arrowheads.

      Furthermore, type V systems also contain a collection of heterogeneous and poorly characterized subtype V‐U; these systems typically lack the adaptation module and contain much smaller effector proteins, making them unlikely to be functional. However, follow‐up studies have identified interference functions with some, prompting their upgrade to a separate type V‐F (Harrington et al. 2018). Finally, an unusual group of what are now Cas12k proteins have been experimentally shown to interact with a bacterial transposase and promote transposon integration through a crRNA‐guided mechanism (Strecker et al. 2019b); this is likely to reflect the proposed evolutionary trajectory of CRISPR systems, proposed to originate as machinery important for transposition (Makarova et al. 2019).

       3.3.2.2.3 Type VI

      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

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