CRISPR-Cas Part II

spinqualitylabelsall models E. coli CRISPR-Cas9 complex with RNA and DNA (PDB code 4qyz) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D SEE CRISPR-Cas Part I CRISPR adaptation The spacers of a CRISPR array represent a chronological archive of previous invader encounters. The captured spacer sequences are integrated into the CRISPR loci after exposure to MGEs, at the leader end of the array that contains the start site of CRISPR transcription. Analysis of invader target sequences (also called protospacers) has revealed a short motif directly adjacent to the target sequence, called the protospacer adjacent motif (PAM). This PAM motif allows self/nonself discrimination by the host in two ways: (i) because its presence in alien targets is required for nonself interference, and (ii) because its absence in the host’s CRISPR array avoids self-targeting. In class 1–type I and class 2–type II systems, the PAM is not only involved in interference, but also plays a role in spacer selection during the adaptation stage, implying the acquisition of functional spacers only. The PAM is a short [2 to 7 nucleotides (nt)], partially redundant sequence that in itself cannot preclude incorporation of spacers from the host DNA because of the low information content of the motif. The short PAM appears to be the result of an evolutionary trade-off between efficient incorporation of spacers from nonself DNA and preventing an autoimmune reaction. Examples of PAM: PAM-complementary dual-forked DNA, which is a 23-mer palindromic duplex from Escherichia coli (5dqz). PAM in crystal structure of Acidaminococcus sp. Cpf1 in complex with crRNA and target DNA (5b43). PAM in crRNA-dsDNA hybrid from E. coli (5h9f). PAM in Cas9-sgRNA-target DNA complex from Streptococcus pyogenes (5fw2). PAM in Cas9-sgRNA-target DNA complex from Francisella tularensis (5b2p). PAM in Cas9-sgRNA-target DNA complex from Staphylococcus aureus (4axw). Few nucleotide long conserved motif recognized directly by Cas9 protein (protospacer adjacent motif, PAM). Although host chromosomal fragments can be incorporated as new CRISPR spacers, detection of such events obviously implies that this did not result in a lethal phenotype, either due to a modified PAM and/or to an inactivated CRISPR-Cas effector module . Indeed, in the absence of the effector module, elevated frequencies of self-spacer acquisition occur in Escherichia coli . Similarly, Streptococcus thermophilus with a catalytically inactive Cas9 results in a major increase of spacers derived from the host genome . In addition, there is a strong preference for the integration of plasmid over chromosomal spacer sequences , with plasmid sequences incorporated more frequently than host DNA by two to three orders of magnitude . Spacer acquisition in E. coli requires active replication of the protospacercontaining DNA . Thus, small, fast-replicating plasmid genomes are a much better source of spacers than the large host DNA, and such findings are consistent with acquisition of spacers from an infecting virus genome in the archaeon Sulfolobus islandicus requiring its active replication . In E. coli, the CRISPR-Cas system derives the spacers primarily from products of RecBCD-catalyzed DNA degradation that are formed during the repair of double-stranded breaks associated with stalled replication forks . Other possible sources of substrates for CRISPR adaptation include DNA fragments generated either by other defense systems, such as restriction-modification systems , or by the CRISPR-Cas system itself . Cas1 and Cas2 play crucial roles in spacer acquisition in all CRISPR-Cas systems . In addition, these proteins can function in trans, provided that the repeats involved are sufficiently similar in size and structure. Accordingly, cas1 and cas2 genes are missing in many active CRISPR-Cas loci—in particular, of type III as well as types IV and VI . Overexpression of Cas1 and Cas2 from the E. coli type I-E system has been shown to be sufficient for the extension of the CRISPR array . Mutations in the active site of Cas1 abolish spacer integration in E. coli , whereas the nuclease activity of Cas2 is dispensable . In E. coli, a central Cas2 dimer and two flanking Cas1 dimers form a complex that binds and processes PAM containing DNA fragments (5dqz; Fig. 3A , ), after which the newly generated spacers can be integrated into a CRISPR array via a recombination mechanism akin to that of retroviral integrases and transposases (Fig. 3B). Cas1 cleaves the phosphodiester bond between nucleotides 28 and 29, resulting in a DNA cleavage product. Glu141, His208, and Asp221 are the catalytic residues of Cas1. The DNA cleavage site is labeled in red. Fig. 3 Spacer acquisition. (A) Crystal structure of the complex of Cas1-Cas2 bound to the dual-forked DNA (PDB accession 5dqz). The target DNA is shown in dark blue; the Cas1 and Cas2 dimers of the complex are indicated in blue and yellow, respectively. (B) Model explaining the capture of new DNA sequences from invading nucleic acid and the subsequent DNA integration into the host CRISPR array. The numbers on the left correspond to the order of events as described in the text. The dashed lines indicate nucleotides; the nucleotides C and N on the two sides of the protospacer are shown in red and green to clarify the orientation. From In several type III CRISPR-Cas systems, Cas1 is fused to reverse transcriptase , and it was recently shown that these systems are capable of acquisition of RNA spacers by direct incorporation of an RNA segment into the CRISPR array followed by reverse transcription and replacement of the RNA strand by DNA . Although the biological function of this process remains to be elucidated, these findings demonstrate remarkable versatility of adaptation pathways. Spacer acquisition (adaptation) in type I systems proceeds along two distinct paths: (i) naïve acquisition, which occurs during an initial infection, and (ii) primed acquisition, when the CRISPR contains a previously integrated spacer that is complementary to the invading DNA . According to the proposed model, naïve spacer adaptation involves five steps (Fig. 3B): 1) Fragmentation of (mainly) invasive nucleic acids by non-Cas systems [e.g., by RecBCD after stalling a replication fork, or by restriction enzymes (restriction-modification systems) ] or by CRISPR-associated nucleases . Although this step may be non-essential, it probably enhances the efficiency of the overall process and its specificity toward invading DNA. 2) Selection of DNA fragments for (proto) spacers by scanning for potential PAMs (after partial target unwinding) by one of the four Cas1 subunits of the Cas1-Cas2 complex . 3) Measuring of the selected protospacer generating fragments of the correct size with 3′ hydroxyl groups by Cas1 nuclease. 4) Nicking of both strands of the leaderproximal repeat of the CRISPR array at the 5′ ends through a direct nucleophilic attack by the generated 3′ OH groups, resulting in covalent links of each of the strands of the newly selected spacer to the single-stranded repeat ends. 5) Second-strand synthesis and ligation of the repeat flanks by a non-Cas repair system . Primed spacer adaptation so far has been demonstrated only in type I systems . This priming mechanism constitutes a positive feedback loop that facilitates the acquisition of new spacers from formerly encountered genetic elements . Priming can occur even with spacers that contain several mismatches, making them incompetent as guides for targeting the cognate foreign DNA . Based on PAM selection, functional spacers are preferentially acquired during naïve adaptation. This initial acquisition event triggers a rapid priming response after subsequent infections. Priming appears to be a major pathway of CRISPR adaptation, at least for some type I systems . Primed adaptation strongly depends on the spacer sequence , and the acquisition efficiency is highest in close proximity to the priming site. In addition, the orientation of newly inserted spacers indicates a strand bias, which is consistentwith the involvement of singlestranded adaption intermediates . According to one proposed model , replication forks in the invader’s DNA are blocked by the Cascade complex bound to the priming crRNA, enabling the RecG helicase and the Cas3 helicase/nuclease proteins to attack the DNA. The ends at the collapsed forks then could be targeted by RecBCD, which provides DNA fragments for new spacer generation . Given that the use of crRNA for priming has much less strict sequence requirements than direct targeting of the invading DNA, priming is a powerful strategy that might have evolved in the course of the host-parasite arms race to reduce the escape by viral mutants, to provide robust resistance against invading DNA, and to enhance self/nonself discrimination. Naïve as well as primed adaptation in the subtype I-F system of Pseudomonas aeruginosa CRISPR-Cas require both the adaptation and the effector module . In the type II-A system, the Cas9-tracrRNA complex and Csn2 are involved in spacer acquisition along with the Cas1-Cas2 complex ; the involvement of Cas9 in adaptation is likely to be a general feature of type II systems. Although the key residues of Cas9 involved in PAM recognition are dispensable for spacer acquisition, they are essential for the incorporation of new spacers with the correct PAM sequence . The involvement of Cas9 in PAM recognition and protospacer selection suggests that in type II systems Cas1 may have lost this role. Similarly, Cas4 that is present in subtypes IA-D and II-B has been proposed to be involved in the CRISPR adaptation process, and this prediction has been validated experimentally for type I-B . Cas4 is absent in the subtype II-C system of Campylobacter jejuni. Nonetheless, a conserved Cas4-like protein found in Campylobacter bacteriophages can activate spacer acquisition to use host DNA as an effective decoy to bacteriophage DNA. Bacteria that acquire self-spacers and escape phage infection must either overcome CRISPR-mediated autoimmunity by loss of the interference functions, leaving them susceptible to foreign DNA invasions, or tolerate changes in gene regulation . Furthermore, in subtypes I-U and V-B, Cas4 is fused to Cas1, which implies cooperation between these proteins during adaptation. In type I-F systems, Cas2 is fused to Cas3 , which suggests a dual role for Cas3 : involvement in adaptation as well as in interference. These findings support the coupling between the adaptation and interference stages of CRISPR-Cas defense during priming. Biogenesis of crRNAs The short mature crRNAs contain spacer sequences, which are the guides that are responsible for the specificity of CRISPR-Cas immunity . They associate with one or more Cas proteins to form effector complexes that target invading MGEs through crRNA:target sequence–specific recognition. The CRISPR arrays are transcribed as long precursors, known as precrRNA, that may contain secondary structured elements (hairpins) in those cases where the CRISPR contains palindromic repeats. The processing of the pre-crRNA typically yields 30- to 65-nt mature crRNAs that consist of a single spacer flanked by a partial repeat at either one or both ends . The pathways of crRNA biogenesis differ among the different CRISPR-Cas types. In class 1 systems, the Cas6 protein is critical for the primary processing of pre-crRNA. Cas6 is a metal-independent endoribonuclease that recognizes and cleaves a single phosphodiester bond in the repeat sequences of a pre-crRNA transcript. Members of the Cas6 family contain two RRM-type RNA-binding domains. The primary cleavage by Cas6 results in crRNAs containing a repeat-derived 5′ “handle” of 8 nt with a 5′ hydroxyl group, followed by the complete spacer sequence and a repeat-derived 3′ handle of variable size that in some subtypes forms a hairpin structure with either a 3′-phosphate or a cyclic 2′,3′-phosphate. The Cas6 family proteins show considerable structural variation that might reflect the cleavage specificity. Summary of the most extensively characterized CRISPR endoribonucleases Representatives of class 1 and class 2 Class 1 CRISPR-Cas systems are considered to be the evolutionary ancestral systems. The class 2 systems have evolved from class 1 systems via the insertion of transposable elements encoding various nucleases, and are now being used as tools for genome editing. Class 1: Cascade complex (Subtype I-E) (4qyz). Cmr complex (Subtype III-B) (3x1l). Class 2: Cas9 complex (Subtype II) (5f9r). Cpf1 complex (Subtype V-A) 5b43. Class 1 CRISPR subtype Orphan TthCas6A (TTHB78) from T. thermophilus. TthCas6A bound to crRNA (4c8z). Other representatives: 4c8y, 4c97. See aslo Cas9 Endonuclease

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