CRISPR-Cas Part II
From Proteopedia
CRISPR adaptationThe 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.[1] Examples of 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 [2]. Indeed, in the absence of the effector module, elevated frequencies of self-spacer acquisition occur in Escherichia coli [3]. Similarly, Streptococcus thermophilus with a catalytically inactive Cas9 results in a major increase of spacers derived from the host genome [4]. In addition, there is a strong preference for the integration of plasmid over chromosomal spacer sequences [3], with plasmid sequences incorporated more frequently than host DNA by two to three orders of magnitude [5]. Spacer acquisition in E. coli requires active replication of the protospacercontaining DNA [5]. 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 [6]. 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 [7]. Other possible sources of substrates for CRISPR adaptation include DNA fragments generated either by other defense systems, such as restriction-modification systems [8], or by the CRISPR-Cas system itself [9].[1] Cas1 and Cas2 play crucial roles in spacer acquisition in all CRISPR-Cas systems [3]. 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 [10]. 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 [3]. Mutations in the active site of Cas1 abolish spacer integration in E. coli [3], whereas the nuclease activity of Cas2 is dispensable [11]. 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 [11], [12]), 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 [13] (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 [1] In several type III CRISPR-Cas systems, Cas1 is fused to reverse transcriptase [14], 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 [15]. 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 [16]. 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) [5][8]] or by CRISPR-associated nucleases [9]. 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 [17]. 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 [18][13].[1] Primed spacer adaptation so far has been demonstrated only in type I systems [19][20][21]. This priming mechanism constitutes a positive feedback loop that facilitates the acquisition of new spacers from formerly encountered genetic elements [22]. Priming can occur even with spacers that contain several mismatches, making them incompetent as guides for targeting the cognate foreign DNA [22]. 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 [20]. Primed adaptation strongly depends on the spacer sequence [23], 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 [24]. According to one proposed model [25], 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 [25]. 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 [24].[1] In the type II-A system, the Cas9-tracrRNA complex and Csn2 are involved in spacer acquisition along with the Cas1-Cas2 complex [4][26]; 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 [26]. The involvement of Cas9 in PAM recognition and protospacer selection [26] 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 [20]. 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 [27]. 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 [28], which suggests a dual role for Cas3 [29]: 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.[1] Biogenesis of crRNAsThe short mature crRNAs contain spacer sequences, which are the guides that are responsible for the specificity of CRISPR-Cas immunity [30]. 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 [30][31].[1] 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.[1] Target interferenceSummary of the most extensively characterized CRISPR endoribonucleases[32][1]Representatives of class 1 and class 2Class 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.[1]
Cascade complex (Subtype I-E) (4qyz). Cmr complex (Subtype III-B) (3x1l).
Cas9 complex (Subtype II) (5f9r). Cpf1 complex (Subtype V-A) 5b43. Class 1CRISPR subtype I-A (Cascade) - SEE CRISPR subtype I-ACRISPR subtype I-B (Cascade) - SEE CRISPR subtype I-BCRISPR subtype I-C (Cascade) - SEE CRISPR subtype I-CCRISPR subtype I-E (Cascade) - SEE CRISPR subtype I-ECRISPR subtype I-F (Cascade) - SEE CRISPR subtype I-FCRISPR subtype III-A (Csm complex) - SEE CRISPR subtype III-A (Csm complex)CRISPR subtype III-B (Cmr complex)CRISPR subtype Orphan
CRISPR type IV (Csf1)Class 2CRISPR type II - SEE CRISPR-Cas9CRISPR type V (Cpf1, C2c1, C2c3) - SEE CRISPR type VCRISPR type VI (Cas13a (previously known as C2c2), Cas13b, Cas13c, Cas13d) - SEE CRISPR type VISee aslo |
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References
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 Mohanraju P, Makarova KS, Zetsche B, Zhang F, Koonin EV, van der Oost J. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science. 2016 Aug 5;353(6299):aad5147. doi: 10.1126/science.aad5147. PMID:27493190 doi:http://dx.doi.org/10.1126/science.aad5147
- ↑ Stern A, Keren L, Wurtzel O, Amitai G, Sorek R. Self-targeting by CRISPR: gene regulation or autoimmunity? Trends Genet. 2010 Aug;26(8):335-40. doi: 10.1016/j.tig.2010.05.008. Epub 2010, Jul 1. PMID:20598393 doi:http://dx.doi.org/10.1016/j.tig.2010.05.008
- ↑ 3.0 3.1 3.2 3.3 3.4 Yosef I, Goren MG, Qimron U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 2012 Jul;40(12):5569-76. doi: 10.1093/nar/gks216. Epub 2012, Mar 8. PMID:22402487 doi:http://dx.doi.org/10.1093/nar/gks216
- ↑ 4.0 4.1 Wei Y, Terns RM, Terns MP. Cas9 function and host genome sampling in Type II-A CRISPR-Cas adaptation. Genes Dev. 2015 Feb 15;29(4):356-61. doi: 10.1101/gad.257550.114. PMID:25691466 doi:http://dx.doi.org/10.1101/gad.257550.114
- ↑ 5.0 5.1 5.2 Levy A, Goren MG, Yosef I, Auster O, Manor M, Amitai G, Edgar R, Qimron U, Sorek R. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature. 2015 Apr 23;520(7548):505-10. doi: 10.1038/nature14302. Epub 2015 Apr 13. PMID:25874675 doi:http://dx.doi.org/10.1038/nature14302
- ↑ Erdmann S, Le Moine Bauer S, Garrett RA. Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus. Mol Microbiol. 2014 Mar;91(5):900-17. doi: 10.1111/mmi.12503. Epub 2014 Jan 17. PMID:24433295 doi:http://dx.doi.org/10.1111/mmi.12503
- ↑ Wigley DB. RecBCD: the supercar of DNA repair. Cell. 2007 Nov 16;131(4):651-3. PMID:18022359 doi:http://dx.doi.org/10.1016/j.cell.2007.11.004
- ↑ 8.0 8.1 Dupuis ME, Villion M, Magadan AH, Moineau S. CRISPR-Cas and restriction-modification systems are compatible and increase phage resistance. Nat Commun. 2013;4:2087. doi: 10.1038/ncomms3087. PMID:23820428 doi:http://dx.doi.org/10.1038/ncomms3087
- ↑ 9.0 9.1 Swarts DC, Mosterd C, van Passel MW, Brouns SJ. CRISPR interference directs strand specific spacer acquisition. PLoS One. 2012;7(4):e35888. doi: 10.1371/journal.pone.0035888. Epub 2012 Apr 27. PMID:22558257 doi:http://dx.doi.org/10.1371/journal.pone.0035888
- ↑ Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV. An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol. 2015 Nov;13(11):722-36. doi: 10.1038/nrmicro3569. Epub 2015, Sep 28. PMID:26411297 doi:http://dx.doi.org/10.1038/nrmicro3569
- ↑ 11.0 11.1 Nunez JK, Kranzusch PJ, Noeske J, Wright AV, Davies CW, Doudna JA. Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity. Nat Struct Mol Biol. 2014 May 4. doi: 10.1038/nsmb.2820. PMID:24793649 doi:http://dx.doi.org/10.1038/nsmb.2820
- ↑ Wang J, Li J, Zhao H, Sheng G, Wang M, Yin M, Wang Y. Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems. Cell. 2015 Nov 5;163(4):840-53. doi: 10.1016/j.cell.2015.10.008. Epub 2015 Oct, 17. PMID:26478180 doi:http://dx.doi.org/10.1016/j.cell.2015.10.008
- ↑ 13.0 13.1 Nunez JK, Lee AS, Engelman A, Doudna JA. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity. Nature. 2015 Mar 12;519(7542):193-8. doi: 10.1038/nature14237. Epub 2015 Feb 18. PMID:25707795 doi:http://dx.doi.org/10.1038/nature14237
- ↑ van der Oost J, Jore MM, Westra ER, Lundgren M, Brouns SJ. CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem Sci. 2009 Aug;34(8):401-7. doi: 10.1016/j.tibs.2009.05.002. Epub, 2009 Jul 29. PMID:19646880 doi:http://dx.doi.org/10.1016/j.tibs.2009.05.002
- ↑ Silas S, Mohr G, Sidote DJ, Markham LM, Sanchez-Amat A, Bhaya D, Lambowitz AM, Fire AZ. Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein. Science. 2016 Feb 26;351(6276):aad4234. doi: 10.1126/science.aad4234. PMID:26917774 doi:http://dx.doi.org/10.1126/science.aad4234
- ↑ Fineran PC, Charpentier E. Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new information. Virology. 2012 Dec 20;434(2):202-9. doi: 10.1016/j.virol.2012.10.003. Epub 2012, Nov 2. PMID:23123013 doi:http://dx.doi.org/10.1016/j.virol.2012.10.003
- ↑ Arslan Z, Hermanns V, Wurm R, Wagner R, Pul U. Detection and characterization of spacer integration intermediates in type I-E CRISPR-Cas system. Nucleic Acids Res. 2014 Jul;42(12):7884-93. doi: 10.1093/nar/gku510. Epub 2014, Jun 11. PMID:24920831 doi:http://dx.doi.org/10.1093/nar/gku510
- ↑ Amitai G, Sorek R. CRISPR-Cas adaptation: insights into the mechanism of action. Nat Rev Microbiol. 2016 Feb;14(2):67-76. doi: 10.1038/nrmicro.2015.14. Epub 2016 , Jan 11. PMID:26751509 doi:http://dx.doi.org/10.1038/nrmicro.2015.14
- ↑ Datsenko KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat Commun. 2012 Jul 10;3:945. doi: 10.1038/ncomms1937. PMID:22781758 doi:http://dx.doi.org/10.1038/ncomms1937
- ↑ 20.0 20.1 20.2 Li M, Wang R, Zhao D, Xiang H. Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process. Nucleic Acids Res. 2014 Feb;42(4):2483-92. doi: 10.1093/nar/gkt1154. Epub 2013, Nov 21. PMID:24265226 doi:http://dx.doi.org/10.1093/nar/gkt1154
- ↑ Richter C, Dy RL, McKenzie RE, Watson BN, Taylor C, Chang JT, McNeil MB, Staals RH, Fineran PC. Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer. Nucleic Acids Res. 2014 Jul;42(13):8516-26. doi: 10.1093/nar/gku527. Epub 2014, Jul 2. PMID:24990370 doi:http://dx.doi.org/10.1093/nar/gku527
- ↑ 22.0 22.1 Fineran PC, Gerritzen MJ, Suarez-Diez M, Kunne T, Boekhorst J, van Hijum SA, Staals RH, Brouns SJ. Degenerate target sites mediate rapid primed CRISPR adaptation. Proc Natl Acad Sci U S A. 2014 Apr 22;111(16):E1629-38. doi:, 10.1073/pnas.1400071111. Epub 2014 Apr 7. PMID:24711427 doi:http://dx.doi.org/10.1073/pnas.1400071111
- ↑ Xue C, Seetharam AS, Musharova O, Severinov K, Brouns SJ, Severin AJ, Sashital DG. CRISPR interference and priming varies with individual spacer sequences. Nucleic Acids Res. 2015 Dec 15;43(22):10831-47. doi: 10.1093/nar/gkv1259. Epub, 2015 Nov 19. PMID:26586800 doi:http://dx.doi.org/10.1093/nar/gkv1259
- ↑ 24.0 24.1 Vorontsova D, Datsenko KA, Medvedeva S, Bondy-Denomy J, Savitskaya EE, Pougach K, Logacheva M, Wiedenheft B, Davidson AR, Severinov K, Semenova E. Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery. Nucleic Acids Res. 2015 Dec 15;43(22):10848-60. doi: 10.1093/nar/gkv1261. Epub, 2015 Nov 19. PMID:26586803 doi:http://dx.doi.org/10.1093/nar/gkv1261
- ↑ 25.0 25.1 Ivancic-Bace I, Cass SD, Wearne SJ, Bolt EL. Different genome stability proteins underpin primed and naive adaptation in E. coli CRISPR-Cas immunity. Nucleic Acids Res. 2015 Dec 15;43(22):10821-30. doi: 10.1093/nar/gkv1213. Epub, 2015 Nov 17. PMID:26578567 doi:http://dx.doi.org/10.1093/nar/gkv1213
- ↑ 26.0 26.1 26.2 Heler R, Samai P, Modell JW, Weiner C, Goldberg GW, Bikard D, Marraffini LA. Cas9 specifies functional viral targets during CRISPR-Cas adaptation. Nature. 2015 Mar 12;519(7542):199-202. doi: 10.1038/nature14245. Epub 2015 Feb, 18. PMID:25707807 doi:http://dx.doi.org/10.1038/nature14245
- ↑ Hooton SP, Connerton IF. Campylobacter jejuni acquire new host-derived CRISPR spacers when in association with bacteriophages harboring a CRISPR-like Cas4 protein. Front Microbiol. 2015 Jan 5;5:744. doi: 10.3389/fmicb.2014.00744. eCollection, 2014. PMID:25601859 doi:http://dx.doi.org/10.3389/fmicb.2014.00744
- ↑ Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct. 2006 Mar 16;1:7. PMID:16545108 doi:http://dx.doi.org/10.1186/1745-6150-1-7
- ↑ van der Oost J, Jore MM, Westra ER, Lundgren M, Brouns SJ. CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem Sci. 2009 Aug;34(8):401-7. doi: 10.1016/j.tibs.2009.05.002. Epub, 2009 Jul 29. PMID:19646880 doi:http://dx.doi.org/10.1016/j.tibs.2009.05.002
- ↑ 30.0 30.1 Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science. 2008 Dec 19;322(5909):1843-5. doi: 10.1126/science.1165771. PMID:19095942 doi:http://dx.doi.org/10.1126/science.1165771
- ↑ Charpentier E, Richter H, van der Oost J, White MF. Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity. FEMS Microbiol Rev. 2015 May;39(3):428-41. doi: 10.1093/femsre/fuv023. Epub 2015 , May 19. PMID:25994611 doi:http://dx.doi.org/10.1093/femsre/fuv023
- ↑ Hochstrasser ML, Doudna JA. Cutting it close: CRISPR-associated endoribonuclease structure and function. Trends Biochem Sci. 2015 Jan;40(1):58-66. doi: 10.1016/j.tibs.2014.10.007. Epub, 2014 Nov 18. PMID:25468820 doi:http://dx.doi.org/10.1016/j.tibs.2014.10.007
Categories: Topic Page | Crispr | Crispr-associated | Endonuclease | Cas9 | Cas6