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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).