Genome Editing in Drug Discovery. Группа авторов
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Название: Genome Editing in Drug Discovery
Автор: Группа авторов
Издательство: John Wiley & Sons Limited
Жанр: Биология
isbn: 9781119671398
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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.
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, СКАЧАТЬ