Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition

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Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.

Vyšlo v časopise: Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition. PLoS Genet 9(9): e32767. doi:10.1371/journal.pgen.1003742
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003742

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Zdroje

1. LabrieSJ, SamsonJE, MoineauS (2010) Bacteriophage resistance mechanisms. Nat Rev Microbiol 8 : 317–327.

2. BikardD, MarraffiniLA (2011) Innate and adaptive immunity in bacteria: mechanisms of programmed genetic variation to fight bacteriophages. Curr Opin Immunol 24 : 15–20.

3. WestraER, SwartsD, StaalsR, JoreM, BrounsSJJ, OostJvd (2012) The CRISPRs they are a-changin'-how prokaryotes generate adaptive immunity. Annu Rev Genet 46 : 311–339.

4. BhayaD, DavisonM, BarrangouR (2011) CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 45 : 273–297.

5. WiedenheftB, SternbergSH, DoudnaJA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482 : 331–338.

6. TernsMP, TernsRM (2011) CRISPR-based adaptive immune systems. Curr Opin Microbiol 14 : 321–327.

7. RichterC, ChangJT, FineranPC (2012) Function and Regulation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated (Cas) Systems. Viruses 4 : 2291–2311.

8. SwartsDC, MosterdC, van PasselMW, BrounsSJ (2012) CRISPR interference directs strand specific spacer acquisition. PloS one 7: e35888.

9. YosefI, GorenMG, QimronU (2012) Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res 40 : 5569–5576.

10. BarrangouR, FremauxC, DeveauH, RichardsM, BoyavalP, et al. (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315 : 1709–1712.

11. DatsenkoKA, PougachK, TikhonovA, WannerBL, SeverinovK, et al. (2012) Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat Commun 3 : 945.

12. MakarovaKS, HaftDH, BarrangouR, BrounsSJ, CharpentierE, et al. (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9 : 467–477.

13. BrounsSJJ, JoreMM, LundgrenM, WestraER, SlijkhuisRJH, et al. (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321 : 960–964.

14. HaurwitzRE, JinekM, WiedenheftB, ZhouK, DoudnaJA (2010) Sequence -⁠ and structure-specific RNA processing by a CRISPR endonuclease. Science 329 : 1355–1358.

15. CarteJ, WangRY, LiH, TernsRM, TernsMP (2008) Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Gene Dev 22 : 3489–3496.

16. PrzybilskiR, RichterC, GristwoodT, ClulowJS, VercoeRB, et al. (2011) Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum. RNA Biol 8 : 517–528.

17. NamKH, HaitjemaC, LiuX, DingF, WangH, et al. (2012) Cas5d protein processes pre-crRNA and assembles into a Cascade-like interference complex in subtype I-C/Dvulg CRISPR-Cas system. Structure 20 : 1574–84.

18. GarsideEL, SchellenbergMJ, GesnerEM, BonannoJB, SauderJM, et al. (2012) Cas5d processes pre-crRNA and is a member of a larger family of CRISPR RNA endonucleases. RNA 18 : 2020–2028.

19. HaleC, KleppeK, TernsRM, TernsMP (2008) Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 14 : 2572–2579.

20. Hatoum-AslanA, ManivI, MarraffiniLA (2011) Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc Natl Acad Sci U S A 108 : 21218–21222.

21. DeltchevaE, ChylinskiK, SharmaCM, GonzalesK, ChaoY, et al. (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471 : 602–607.

22. JoreMM, LundgrenM, van DuijnE, BultemaJB, WestraER, et al. (2011) Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol 18 : 529–536.

23. HaleCR, ZhaoP, OlsonS, DuffMO, GraveleyBR, et al. (2009) RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139 : 945–956.

24. ZhangJ, RouillonC, KerouM, ReeksJ, BruggerK, et al. (2012) Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol Cell 45 : 303–313.

25. LintnerNG, KerouM, BrumfieldSK, GrahamS, LiuH, et al. (2011) Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE). J Biol Chem 286 : 21643–21656.

26. WiedenheftB, van DuijnE, BultemaJB, WaghmareSP, ZhouK, et al. (2011) RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci U S A 108 : 10092–10097.

27. JinekM, ChylinskiK, FonfaraI, HauerM, DoudnaJA, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337 : 816–821.

28. HaleCR, MajumdarS, ElmoreJ, PfisterN, ComptonM, et al. (2012) Essential features and rational design of CRISPR RNAs that function with the Cas RAMP Module Complex to cleave RNAs. Mol Cell 45 : 292–302.

29. WestraER, van ErpPB, KunneT, WongSP, StaalsRH, et al. (2012) CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol Cell 46 : 595–605.

30. GarneauJE, DupuisME, VillionM, RomeroDA, BarrangouR, et al. (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468 : 67–71.

31. ManicaA, ZebecZ, TeichmannD, SchleperC (2011) In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon. Mol Microbiol 80 : 481–491.

32. MarraffiniLA, SontheimerEJ (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322 : 1843–1845.

33. EdgarR, QimronU (2010) The Escherichia coli CRISPR system protects from lambda lysogenization, lysogens, and prophage induction. J Bacteriol 192 : 6291–6294.

34. SternA, KerenL, WurtzelO, AmitaiG, SorekR (2010) Self-targeting by CRISPR: gene regulation or autoimmunity? Trends Genet 26 : 335–340.

35. VercoeRB, ChangJT, DyRL, TaylorC, GristwoodT, et al. (2013) Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet 9: e1003454.

36. MarraffiniLA, SontheimerEJ (2010) Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463 : 568–571.

37. KuninV, SorekR, HugenholtzP (2007) Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol 8: R61.

38. MojicaFJM, Diez-VillasenorC, Garcia-MartinezJ, AlmendrosC (2009) Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155 : 733–740.

39. FischerS, MaierLK, StollB, BrendelJ, FischerE, et al. (2012) An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA. J Biol Chem 287 : 33351–33363.

40. GudbergsdottirS, DengL, ChenZ, JensenJV, JensenLR, et al. (2011) Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers. Mol Microbiol 79 : 35–49.

41. SemenovaE, JoreMM, DatsenkoKA, SemenovaA, WestraER, et al. (2011) Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A 108 : 10098–10103.

42. GorenMG, YosefI, AusterO, QimronU (2012) Experimental definition of a Clustered Regularly Interspaced Short Palindromic duplicon in Escherichia coli. J Mol Biol 423 : 14–16.

43. AlmendrosC, GuzmanNM, Diez-VillasenorC, Garcia-MartinezJ, MojicaFJ (2012) Target motifs affecting natural immunity by a constitutive CRISPR-Cas system in Escherichia coli. PLoS One 7: e50797.

44. SashitalDG, WiedenheftB, DoudnaJA (2012) Mechanism of foreign DNA selection in a bacterial adaptive immune system. Mol Cell 46 : 606–615.

45. SinkunasT, GasiunasG, WaghmareSP, DickmanMJ, BarrangouR, et al. (2013) In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus. EMBO J 32 : 385–394.

46. Dupuis M, Moineau S (2013) Type II: Streptococcus thermophilus. In: Barrangou R, van der Oost J, editors. CRISPR-Cas Systems -⁠ RNA-mediated Adaptive Immunity in Bacteria and Archaea: Springer. pp. 171–200.

47. WiedenheftB, LanderGC, ZhouK, JoreMM, BrounsSJ, et al. (2011) Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477 : 486–489.

48. ErdmannS, GarrettRA (2012) Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms. Mol Microbiol 85 : 1044–1056.

49. WestraER, BrounsSJ (2012) The rise and fall of CRISPRs -⁠ dynamics of spacer acquisition and loss. Mol Microbiol 85 : 1021–1025.

50. Lopez-SanchezMJ, SauvageE, Da CunhaV, ClermontD, Ratsima HariniainaE, et al. (2012) The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome. Mol Microbiol 85 : 1057–1071.

51. ShahSA, ErdmannS, MojicaFJ, GarrettRA (2013) Protospacer recognition motifs: mixed identities and functional diversity. RNA biol 10 : 891–899.

52. CadyKC, Bondy-DenomyJ, HeusslerGE, DavidsonAR, O'TooleGA (2012) The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages. J Bacteriol 194 : 5728–5738.

53. DeveauH, BarrangouR, GarneauJE, LabonteJ, FremauxC, et al. (2008) Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 190 : 1390–1400.

54. MagadanAH, DupuisME, VillionM, MoineauS (2012) Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system. PLoS One 7: e40913.

55. MulepatiS, OrrA, BaileyS (2012) Crystal structure of the largest subunit of a bacterial RNA-guided immune complex and its role in DNA target binding. J Biol Chem 287 : 22445–22449.

56. MakarovaKS, AravindL, WolfYI, KooninEV (2011) Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct 6 : 38.

57. PulU, WurmR, ArslanZ, GeissenR, HofmannN, et al. (2010) Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol Microbiol 75 : 1495–1512.

58. WestraER, PulU, HeidrichN, JoreMM, LundgrenM, et al. (2010) H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO. Mol Microbiol 77 : 1380–1393.

59. PougachK, SemenovaE, BogdanovaE, DatsenkoKA, DjordjevicM, et al. (2010) Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol Microbiol 77 : 1367–1379.

60. MillenAM, HorvathP, BoyavalP, RomeroDA (2012) Mobile CRISPR/Cas-mediated bacteriophage resistance in Lactococcus lactis. PLoS One 7: e51663.