Genome Editing in Drug Discovery. Группа авторов
Читать онлайн книгу.as well; however, the intricacies and finesses of further classification are beyond the scope of this Chapter and the reader is invited to consult recent excellent reviews on the topic (Hille et al. 2018; Koonin and Makarova 2019; Makarova et al. 2019; Nussenzweig and Marraffini 2020).
3.3.1 crRNA Biogenesis
Synthesis of crRNAs starts with the transcription of the CRISPR array from a promoter usually located within the leader sequence (Pul et al. 2010; Pougach et al. 2010). Processing of the pre‐crRNA is specific to each of the CRISPR class, with class 1 pre‐crRNAs cleaved by dedicated endoribonuclease, whereas the class 2 systems employ the same machinery that performs target destruction (Figure 3.3).
In class 1 systems, the processing is performed by either Cas6 or Cas5d ribonucleases (Nam et al. 2012; Carte et al. 2008). Both of these proteins recognize and bind to the hairpin structure formed by the palindromic sequences of the pre‐cRNA, and introduce a cut immediately downstream of it (Figure 3.3a), releasing mature crRNAs (Carte et al. 2010; Haurwitz et al. 2010; Ozcan et al. 2019). Intriguingly, in CRISPR systems containing repeats which are not thermodynamically likely to form hairpin structures (namely type I‐A and ‐B, and type III‐A and ‐B) and, hence, lack inherent discriminatory borders between spacers, Cas6 seems to be able to identify repeat regions by restructuring them to favor the formation of a hairpin or hairpin‐like structure compatible with precise cleavage that will lead to productive crRNAs (Shao et al. 2016; Sefcikova et al. 2017). Mature crRNAs of most type I systems contain part of the repeat sequence at the 5’ end of the spacer and the 3’ hairpin; these do not participate in recognition of the target sequence but seem to be important for the assembly of the effector complex (Jore et al. 2011). Type III crRNA, on the other hand, undergoes additional trimming that removes the hairpin structure (Hale et al. 2008). How these mature crRNAs are paired to the cognate effector complex remains unanswered.
Class 2 systems employ two different strategies to generate mature crRNAs. The first strategy employed by type V and VI is in principle similar to class 1 crRNA biogenesis (Figure 3.3b). Here, the effector nucleases, such as Cas12a (Cpf1) and Cas13, recognize the repeat hairpin structure within the pre‐crRNA and cleave the RNA within or upstream of it (East‐Seletsky et al. 2016; Fonfara et al. 2016).
A more elaborate strategy is used to generate mature crRNAs in type II and type V‐B systems (Figure 3.3c). These two systems, exemplified by Cas9 and Cas12b (C2c1), require a second noncoding trans‐activating CRISPR RNA (tracrRNA) to pair with the repeat regions within the pre‐crRNA and form an intermediary between the crRNA and the effector protein (Deltcheva et al. 2011; Shmakov et al. 2015). The stem‐loops of the tracrRNA act as a recruitment site for Cas9 and Cas12b, permitting them to form a ternary pre‐crRNA:tracrRNA:Cas effector complex. The binding of the effector protein further stabilizes the interaction between pre‐crRNA and tracrRNA, but also recruits cellular RNase III that cleaves the RNA:RNA duplex formed by the repeat sequences of the pre‐crRNA and tracrRNA, releasing the 3’ end of the crRNA (Deltcheva et al. 2011). The 5’ end of the crRNA is processed further by removing the remaining repeat sequence, but the protein involved has remained elusive (Hille et al. 2018; Nussenzweig and Marraffini 2020). Once fully processed, crRNA paired with its cognate effector protein can patrol the cytosol and confer immunity to any invading DNA.
Figure 3.2 Overview of class 1 and class 2 CRISPR systems. General composition of various CRISPR systems across two classes and six types. The top panel shows a legend with a simplified, hypothetical CRISPR system containing key functional modules used in the immune response (adaptation, expression, interference, or ancillary). Functions of homologous genes are distinguished by color and demarcated by shaded areas. Many Cas proteins perform multiple activities in the immune response, and are as such highlighted as multicolored fusions spanning several categories. Many components of CRISPR systems are absent from specific subtypes, and are therefore represented in washout and with a dashed outline and an asterisk. Depicted CRISPR loci are schematic and do not correspond to real‐life examples; hence the gene order, size, and orientation are purely didactic. Nomenclature and general module organization are from the most recent classification (Makarova et al., 2019).
3.3.2 Interference
3.3.2.1 Class 1
3.3.2.1.1 Type I
Type I systems are the most widespread CRISPR‐Cas systems, and were the first to be identified (Ishino et al. 1987) and subsequently characterized. The effector complex of type I systems consists of the complex termed Cascade (CRISPR‐associated complex for antiviral defense) that binds to crRNA, and the signature Cas3 protein which exhibits helicase and nuclease activity (Brouns et al. 2008). The assembly of the Cascade complex initiates after the pre‐crRNA processing catalyzed by the RNase activity of Cas6 protein (Figure 3.4a), which remains bound to the 3’ end of the crRNA (Sashital et al. 2011). Cas6‐crRNA acts as a nucleation center for the assembly of the heterododecameric Cascade complex ((Cas5)1‐(Cas6)1‐(Cas7)6‐(Cas8)1‐(Cas11)2), with Cas7 proteins binding to spacer sequence (inducing a kink at every sixth nucleotide of the crRNA) and Cas5 capping the 5’ end of the crRNA (Mulepati et al. 2014). Cascade complex uses its Cas8 subunit to identify PAM sequence via minor‐groove interactions. Successful recognition of PAM site leads to conformational change of the Cascade complex, which in turn unwinds PAM site and permits pairing of the bound crRNA with the target DNA strand (Figure 3.4a), forming a thermodynamically stable R‐loop that stabilizes the Cascade complex onto the target DNA (Hayes et al. 2016; Xiao et al. 2017). As a consequence of binding to DNA, Cascade is able to recruit Cas3, which makes a single‐stranded break on the nontargeted DNA strand. The subsequent activation of the helicase domain allows Cas3 to translocate on the nontarget strand in 3’–5’ direction, generating 200–300 nt single‐stranded DNA gap (Sinkunas et al. 2011; Sinkunas et al. 2013; Huo et al. 2014; Redding et al. 2015). While generating gaps is not likely to be sufficient to fully destroy the invading DNA, it is likely that the DNA can be degraded by one of the host’s nucleolytic machinery (Lovett 2011).
Figure 3.3 crRNA biogenesis pathways. (a) depicts a canonical crRNA biogenesis pathway for class 1 CRISPR systems, where Cas6 (or Cas5d in some subtypes) RNase recognizes and binds to hairpin structures in the pre‐crRNA, cleaving in their vicinity to liberate mature crRNAs. Cas6 remains bound to crRNA and acts as a platform for the assembly of the rest of Cascade effector complex. Two different strategies for the maturation of crRNAs in class 2 systems are shown in panels b and c. In (b), some class 2 systems (represented here with Cas12a), the effector enzyme itself recognizes the repeats in pre‐crRNA and cleaves the RNA 4 nucleotides upstream of the adjacent hairpin. crRNA processed in such a way remain paired with its Cas enzyme, forming a functional effector complex. In (c), type II systems crRNA processing requires tracrRNA which pairs with repeat regions and subsequently recruit Cas proteins and deploy cellular RNase III that liberates Cas9:crRNA:tracrRNA complex. 5’ end of crRNA is further processed by an unknown cellular nuclease. Colored rectangles represent spacers, gray stem‐loops repeat sequences, and red triangles cleavage sites.