system (Pyenson et al. 2017).
3.3.2.1.3 Type IV
Type IV systems are the most enigmatic category of CRISPR systems. Type IV systems are most frequently found on plasmids, conjugative plasmid elements, and seldom in phage genomes (Pinilla‐Redondo et al. 2020), and typically lack genes involved in adaptation (Cas1, Cas2, and Cas4) or nucleolytic degradation Figure 3.2, and frequently even without the CRISPR arrays (Makarova et al. 2019). Subtype IV‐C, a recently discovered CRISPR system of Thermoflexia bacterium, contains a large subunit (LS or csf1) gene with putative HD nuclease domain, providing the only suggestion that some of the type IV systems might retain nuclease activity (Makarova et al. 2019). Recently, a striking bias toward plasmid sequences as spacers (in contrast to other systems, which predominantly share homology with viral genomes) has suggested that type IV might have a role in plasmid maintenance and competition (Faure et al. 2019a; Newire et al. 2020; Pinilla‐Redondo et al. 2020), whether this is true remains to be experimentally established.
Bioinformatic analyses have observed that type IV systems are frequently found together with type I systems, indicating a potential crosstalk between the two systems. Indeed, PAM, repeat, and leader sequences are nearly identical between the co‐occurring systems (Pinilla‐Redondo et al. 2020), suggesting that type IV systems can be viewed as minimal Cas systems that can rely on the adaptation machinery from “helper” type I system. Recently, a structure of type IV effector complex has revealed a sea cucumber‐like structure, with seven Cas7‐like subunits forming the backbone and interacting with crRNA (inducing a kink in the RNA every sixth nucleotide much like type I Cascade), with five Cas11 forming the belly of the structure (Zhou et al. 2020). Cas6 protein in type IV systems is responsible for processing and binding to crRNA (Ozcan et al. 2019), much like in type I. However, when components of the Mycobacterium sp. type IV system were co‐expressed in E. coli, it was found that crRNA originated from a wide range of sources, including from the plasmid harboring the type IV system, but also from rRNA, tRNA, and other noncoding small RNA. This suggests that there might be indeed crosstalk between the hosts and plasmid‐borne CRISPR systems; however, the apparent lack of specificity in processing/assembling crRNA–effector complex does not support a role in nuclease‐mediated immunity. The fact that type IV systems are frequently accompanied by genes with a potential role in other types of bacterial defense systems, such as cysH or ART gene, supports the notion that this type of system has been co‐opted for plasmid or phage maintenance, rather than their depletion (Faure et al. 2019b). Further studies are needed to unravel the role (if any) of these systems.
3.3.2.2 Class 2
When compared with class 1 systems, class 2 systems are much simpler in the sense that they comprise of a smaller number of components. Class 2 systems all have in common that the effector module is contained within a single protein, in contrast to multiple subunits of class 1 systems discussed so far. This class can be subcategorized based on the key genes involved in interference (Cas9, Cas12, and Cas13), all of which restrict the invading genomes in different ways. While this might not be optimal for a robust immune response, in particular when compared with type III systems, their simplicity makes them a better tool for genome editing purposes. While some class 1 systems have been used for gene editing, most notably in prokaryotes (Kiro et al. 2014; Li et al. 2016; Pyne et al. 2016), thanks to their simplicity (need for only one Cas protein) class 2 systems have been the easiest systems to be adapted for genome engineering. The many interesting activities of class 2 systems will be addressed below.
3.3.2.2.1 Type II
The most well‐known example of type II systems, and arguably of all CRISPR systems, is the S. pyogenes systems, epitomized by Cas9 (abbreviated as SpyCas9) for its use in gene editing in eukaryotic systems (Cong et al. 2013; DiCarlo et al. 2013; Ding et al. 2013; Friedland et al. 2013; Hwang et al. 2013; Jinek et al. 2013; Mali et al. 2013). Cas9 is a dual RNA‐guided DNA endonuclease required for conferring immunity in type II systems (Barrangou et al. 2007; Gasiunas et al. 2012; Jinek et al. 2012). Apart from crRNA (required by any other CRISPR system to recognize target molecule), Cas9 also requires trans‐activating 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).
