Читать книгу Genome Editing in Drug Discovery - Группа авторов - Страница 40

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).