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Figure 3.4 Interference mechanism in class 1 systems. Panel (a) depicts interference in type I systems. Cascade complex assembles on Cas6:crRNA (originating from crRNA biogenesis) and identifies target sequence by recognizing PAM by Cas8 subunit and protospacer by stepwise pairing with the crRNA. Successful pairing allows a Cas3 nuclease and helicase to be recruited. Cas3 unwinds and translocates on the nontarget strand while simultaneously cleaving the same strand (depicted by orange arrows), generating single‐stranded gaps. The interference mechanism typical for type III systems is depicted on panel (b), exemplified by Cas10‐Csm complex. The effector complex assembles with hairpinless crRNA and is targeted to sites of active transcription by hybridizing the crRNA with complementary sequence in nascent RNA. Successful pairing activates RNase (red arrow heads) and nuclease activities of HD domain of Cas10, leading to on‐target degradation of both the transcript and the genomic locus. Activated Cas10 is also able to convert ATP to cyclic oligoadenylates (coA), which stimulate indiscriminate RNase activity of Csm6, leading to global depletion of the cellular transcriptome.

       3.3.2.1.2 Type III

      Type III CRISPR‐Cas systems are based on the Cascade‐like complexes (Csm or Cmr in subtypes III‐A and III‐B, respectively) working with their cognate hairpinless crRNAs. These complexes display overall structural similarity to Cascade complex of type I, both forming a seahorse‐shaped complex (Osawa et al. 2015; Taylor et al. 2015). In the Csm/Cmr complex, the 5’ end of the crRNA is bound by Cas5, with the backbone of multiple proteins belonging to Cas7 (Csm3/Csm5 and Cmr4/Cmr6/Cmr1) and Cas11 (Csm2/Cmr5) protein families. The effector complex is completed by binding of Cas10 protein (Figure 3.4b). The distinct feature of type III systems is that they can degrade both DNA and RNA, through concerted DNase activity of Cas10 and RNase activities of Csm3 and ancillary Csm6 proteins (Hale et al. 2009; Deng et al. 2013; Staals et al. 2014; Samai et al. 2015).

      Type III systems confer immunity against DNA bacteriophages or plasmids (Marraffini and Sontheimer 2008; Hatoum‐Aslan et al. 2014; Samai et al. 2015), but can only target the invading genome if it is actively transcribed (Deng et al. 2013; Goldberg et al. 2014). Whether Csm/Cmr complexes get recruited directly to transcribing RNA polymerase, nascent transcript, or underwound DNA generated in the wake of RNA polymerase remains a contentious topic (Elmore et al. 2016; Han et al. 2017; Liu et al. 2019b). It is clear that interference begins by the pairing of loaded crRNA to complementary nascent transcript (Figure 3.4b), stimulating the nucleolytic degradation of the nontarget DNA strand by Cas10 (Estrella et al. 2016; Liu et al. 2017d). In parallel, Cas7 subunits cleave the paired RNA at every sixth nucleotide (Tamulaitis et al. 2014; Liu et al. 2017d), and in some systems the ancillary RNase Csm6 degrades proximal RNA in an unspecific manner (Jiang et al. 2016b); this dual action of two main nucleases efficiently silences phage RNA and simultaneously disrupts the invasive genome (Figure 3.4b). Type III‐A system have evolved even stronger adaptive response, where targeted binding to RNA stimulates unspecific cleavage of ssDNA by the HD domain of Cas10 (Kazlauskiene et al. 2016; Liu et al. 2017d), and the conversion of ATP to cycling oligoadenylates by its Palm polymerase domain. The cyclic oligoadenylates further stimulate the activity of the HEPN domain of the Csm6 ribonuclease (Kazlauskiene et al. 2017; Niewoehner et al. 2017), leading to an indiscriminate degradation of both host and invading RNA, causing a growth arrest which restricts invader propagation (Jiang et al. 2016b; Rostol and Marraffini 2019). Together, this gives rise to a potent, highly adaptable, and robust immune 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

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