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Analysis of CRISPR Pre-crRNA Cleavage

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CRISPR

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1311))

Abstract

We have examined the processing of precursor-clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (pre-crRNAs) of the Type I CRISPR-Cas system by incubation of radiolabeled model RNAs with recombinant CRISPR-associated (Cas) endoribonucleases, followed by denaturing polyacrylamide gel electrophoresis (PAGE) of the products. Determination of cleavage position is based on comparison with RNase T1 digestion and base hydrolysis products. The mechanism of cleavage is investigated by chemical and enzymatic characterization of the reaction products as well as by the demonstration that a specific 2′-deoxy substitution 5′ to the scissile phosphate blocks endonucleolytic cleavage.

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References

  1. Barrangou R, Fremaux C, Deveau H et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712

    Article  CAS  PubMed  Google Scholar 

  2. Brouns SJJ, Jore MM, Lundgren M et al (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964

    Article  CAS  PubMed  Google Scholar 

  3. Carte J, Wang R, Li H et al (2008) Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev 22:3489–3496

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Jansen R, Embden JD, Gaastra W et al (2002) Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 43:1565–1575

    Article  CAS  PubMed  Google Scholar 

  5. Marraffini LA, Sontheimer EJ (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322:1843–1845

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Haft DH, Selengut J, Mongodin EF et al (2005) A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol 1:e60

    Article  PubMed Central  PubMed  Google Scholar 

  7. Kunin V, Sorek R, Hugenholtz P (2007) Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol 8:R61

    Article  PubMed Central  PubMed  Google Scholar 

  8. Makarova KS, Grishin N, Shabalina S et al (2006) A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzyme machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 1:7–33

    Article  PubMed Central  PubMed  Google Scholar 

  9. Makarova KS, Haft DH, Barrangou R et al (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9:467–477

    Article  CAS  PubMed  Google Scholar 

  10. Gesner E, Schellenberg MJ, Garside EL et al (2011) Recognition and maturation of effector RNAs in a CRISPR interference pathway. Nat Struct Mol Biol 18:688–692

    Article  CAS  PubMed  Google Scholar 

  11. Garside EL, Schellenberg MJ, Gesner EM et al (2012) Cas5d processes pre-crRNA and is a member of a larger family of CRISPR RNA endonucleases. RNA 18:2020–2028

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Makarova KS, Aravind L, Wolf YI et al (2011) Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct 6:38

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Sashital DG, Jinek M, Dounda JA (2011) An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3. Nat Struct Mol Biol 18:680–687

    Article  CAS  PubMed  Google Scholar 

  14. Haurwitz RE, Jinek M, Wiedenheft B et al (2010) Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329:1355–1358

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Carte J, Pfister NT, Compton MM et al (2010) Binding and cleavage of CRISPR RNA by Cas6. RNA 16:2181–2188

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Haurwitz RE, Sternberg SH, Ja D (2012) Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA. EMBO J 31:2824–2832

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Koo Y, Ka D, Kim EJ et al (2013) Conservation and variability in the structure and function of the Cas5d endoribonuclease in the CRISPR-mediated microbial immune system. J Mol Biol 425:3799–3810

    Article  CAS  PubMed  Google Scholar 

  18. Nam KH, Haitjema C, Liu X et al (2012) Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in subtype I-C/DvulgCRISPR-Cas system. Structure 20:1574–1584

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Niewoehner O, Jinek M, Doudna JA (2013) Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases. Nucleic Acids Res 42:1341–1353

    Article  PubMed Central  PubMed  Google Scholar 

  20. Plagens A, Tjaden B, Hagemann A et al (2012) Characterization of the CRISPR/Cas subtype I-A system of the hyperthermophilic crenarchaeon Thermoproteus tenax. J Bacteriol 194:2491–2500

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Przybilski R, Richter C, Gristwood T et al (2011) Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum. RNA Biol 8:517–528

    Article  CAS  PubMed  Google Scholar 

  22. Reeks J, Sokolowski RD, Graham S et al (2013) Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing. Biochem J 452:223–230

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Richter J, Zoephel J, Schermuly J et al (2012) Characterization of CRISPR RNA processing in Clostridium thermocellum and Methanococcus maripaludis. Nucleic Acids Res 40:9887–9896

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Shao Y, Li H (2013) Recognition and cleavage of a nonstructured CRISPR RNA by its processing endoribonuclease Cas6. Structure 21:385–393

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Sternberg SH, Haurwitz RE, Dounda JA (2012) Mechanism of substrate selection by a highly specific CRISPR endoribonuclease. RNA 18:661–672

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Wang R, Preamplume G, Terns MP et al (2011) Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage. Structure 19:257–264

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Wang R, Zheng H, Preamplume G et al (2012) The impact of CRISPR repeat sequence of structures of a Cas6 protein-RNA complex. Protein Sci 21:405–417

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Rio DC, Hannon GJ, Ares M Jr, Nilsen TW (2011) Labeling of oligonucleotide probes (DNA, LNA, RNA) by polynucleotide kinase and [ɣ-32P]ATP. In: RNA: a laboratory manual. Cold Spring Harbor, New York

    Google Scholar 

  29. Rio DC, Hannon GJ, Ares M Jr, Nilsen TW (2011) Gel purification of RNA. In: RNA: a laboratory manual. Cold Spring Harbor, New York

    Google Scholar 

  30. Rio DC, Hannon GJ, Ares M Jr, Nilsen TW (2011) Gel electrophoresis. In: RNA: a laboratory manual. Cold Spring Harbor, New York

    Google Scholar 

  31. Igloi GL, Kossel H (1985) Affinity electrophoresis for monitoring terminal phosphorylation and the presence of queuosine in RNA. Application of polyacrylamide containing a covalently bound boronic acid. Nucleic Acids Res 13:6881–6898

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Biochemistry, University of Alberta, 443 Medical Sciences Building, Edmonton, AB, Canada, T6G 2H7

    Erin L. Garside & Andrew M. MacMillan

Authors
  1. Erin L. Garside
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  2. Andrew M. MacMillan
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Corresponding author

Correspondence to Andrew M. MacMillan .

Editor information

Editors and Affiliations

  1. Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

    Magnus Lundgren

  2. Department of Molecular Biology MIMS, Umeå University, Umeå, Sweden

    Emmanuelle Charpentier

  3. Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

    Peter C. Fineran

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Garside, E.L., MacMillan, A.M. (2015). Analysis of CRISPR Pre-crRNA Cleavage. In: Lundgren, M., Charpentier, E., Fineran, P. (eds) CRISPR. Methods in Molecular Biology, vol 1311. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2687-9_3

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  • DOI: https://doi.org/10.1007/978-1-4939-2687-9_3

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2686-2

  • Online ISBN: 978-1-4939-2687-9

  • eBook Packages: Springer Protocols

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