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Investigating CRISPR RNA Biogenesis and Function Using RNA-seq

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CRISPR

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

Abstract

The development of deep sequencing technology has greatly facilitated transcriptome analyses of both prokaryotes and eukaryotes. RNA-sequencing (RNA-seq), which is based on massively parallel sequencing of cDNAs, has been used to annotate transcript boundaries and revealed widespread antisense transcription as well as a wealth of novel noncoding transcripts in many bacteria. Moreover, RNA-seq is nowadays widely used for gene expression profiling and about to replace hybridization-based approaches such as microarrays. RNA-seq has also informed about the biogenesis and function of CRISPR RNAs (crRNAs) of different types of bacterial RNA-based CRISPR-Cas immune systems. Here we describe several studies that employed RNA-seq for crRNA analyses, with a particular focus on a differential RNA-seq (dRNA-seq) approach, which can distinguish between primary and processed transcripts and allows for a genome-wide annotation of transcriptional start sites. This approach helped to identify a new crRNA biogenesis pathway of Type II CRISPR-Cas systems that involves a trans-encoded small RNA, tracrRNA, and the host factor RNase III.

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References

  1. Mutz KO, Heilkenbrinker A, Lonne M, Walter JG, Stahl F (2013) Transcriptome analysis using next-generation sequencing. Curr Opin Biotechnol 24:22–30

    Article  CAS  PubMed  Google Scholar 

  2. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Croucher NJ, Thomson NR (2010) Studying bacterial transcriptomes using RNA-seq. Curr Opin Microbiol 13:619–624

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. van Vliet AH (2010) Next generation sequencing of microbial transcriptomes: challenges and opportunities. FEMS Microbiol Lett 302:1–7

    Article  PubMed  Google Scholar 

  5. Sorek R, Cossart P (2010) Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity. Nat Rev Genet 11:9–16

    Article  CAS  PubMed  Google Scholar 

  6. Sittka A, Lucchini S, Papenfort K, Sharma CM, Rolle K, Binnewies TT, Hinton JC, Vogel J (2008) Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq. PLoS Genet 4:e1000163

    Article  PubMed Central  PubMed  Google Scholar 

  7. Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S, Sittka A, Chabas S, Reiche K, Hackermuller J, Reinhardt R, Stadler PF, Vogel J (2010) The primary transcriptome of the major human pathogen Helicobacter pylori. Nature 464:250–255

    Article  CAS  PubMed  Google Scholar 

  8. McGrath PT, Lee H, Zhang L, Iniesta AA, Hottes AK, Tan MH, Hillson NJ, Hu P, Shapiro L, McAdams HH (2007) High-throughput identification of transcription start sites, conserved promoter motifs and predicted regulons. Nat Biotechnol 25:584–592

    Article  CAS  PubMed  Google Scholar 

  9. Croucher NJ, Fookes MC, Perkins TT, Turner DJ, Marguerat SB, Keane T, Quail MA, He M, Assefa S, Bahler J, Kingsley RA, Parkhill J, Bentley SD, Dougan G, Thomson NR (2009) A simple method for directional transcriptome sequencing using Illumina technology. Nucleic Acids Res 37:e148

    Article  PubMed Central  PubMed  Google Scholar 

  10. Cloonan N, Forrest AR, Kolle G, Gardiner BB, Faulkner GJ, Brown MK, Taylor DF, Steptoe AL, Wani S, Bethel G, Robertson AJ, Perkins AC, Bruce SJ, Lee CC, Ranade SS, Peckham HE, Manning JM, McKernan KJ, Grimmond SM (2008) Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat Methods 5:613–619

    Article  CAS  PubMed  Google Scholar 

  11. He Y, Vogelstein B, Velculescu VE, Papadopoulos N, Kinzler KW (2008) The antisense transcriptomes of human cells. Science 322:1855–1857

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Parkhomchuk D, Borodina T, Amstislavskiy V, Banaru M, Hallen L, Krobitsch S, Lehrach H, Soldatov A (2009) Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res 37:e123

    Article  PubMed Central  PubMed  Google Scholar 

  13. Frias-Lopez J, Shi Y, Tyson GW, Coleman ML, Schuster SC, Chisholm SW, Delong EF (2008) Microbial community gene expression in ocean surface waters. Proc Natl Acad Sci U S A 105:3805–3810

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Raabe CA, Tang TH, Brosius J, Rozhdestvensky TS (2014) Biases in small RNA deep sequencing data. Nucleic Acids Res 42:1414–1426

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. t Hoen PA, Friedlander MR, Almlof J, Sammeth M, Pulyakhina I, Anvar SY, Laros JF, Buermans HP, Karlberg O, Brannvall M, den Dunnen JT, van Ommen GJ, Gut IG, Guigo R, Estivill X, Syvanen AC, Dermitzakis ET, Lappalainen T (2013) Reproducibility of high-throughput mRNA and small RNA sequencing across laboratories. Nat Biotechnol 31:1015–1022

    Article  CAS  Google Scholar 

  16. Thompson JA, Radonovich MF, Salzman NP (1979) Characterization of the 5′-terminal structure of simian virus 40 early mRNA’s. J Virol 31:437–446

    PubMed Central  CAS  PubMed  Google Scholar 

  17. Argaman L, Hershberg R, Vogel J, Bejerano G, Wagner EG, Margalit H, Altuvia S (2001) Novel small RNA-encoding genes in the intergenic regions of Escherichia coli. Curr Biol 11:941–950

    Article  CAS  PubMed  Google Scholar 

  18. Vogel J, Bartels V, Tang TH, Churakov G, Slagter-Jager JG, Huttenhofer A, Wagner EG (2003) RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria. Nucleic Acids Res 31:6435–6443

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Bensing BA, Meyer BJ, Dunny GM (1996) Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis. Proc Natl Acad Sci U S A 93:7794–7799

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Wurtzel O, Sapra R, Chen F, Zhu Y, Simmons BA, Sorek R (2010) A single-base resolution map of an archaeal transcriptome. Genome Res 20:133–141

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Mendoza-Vargas A, Olvera L, Olvera M, Grande R, Vega-Alvarado L, Taboada B, Jimenez-Jacinto V, Salgado H, Juarez K, Contreras-Moreira B, Huerta AM, Collado-Vides J, Morett E (2009) Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli. PLoS One 4:e7526

    Article  PubMed Central  PubMed  Google Scholar 

  22. Cho BK, Zengler K, Qiu Y, Park YS, Knight EM, Barrett CL, Gao Y, Palsson BO (2009) The transcription unit architecture of the Escherichia coli genome. Nat Biotechnol 27:1043–1049

    Article  CAS  PubMed  Google Scholar 

  23. Sharma CM, Vogel J (2014) Differential RNA-seq: the approach behind and the biological insight gained. Curr Opin Microbiol 19:97–105

    Article  CAS  PubMed  Google Scholar 

  24. Juranek S, Eban T, Altuvia Y, Brown M, Morozov P, Tuschl T, Margalit H (2012) A genome-wide view of the expression and processing patterns of Thermus thermophilus HB8 CRISPR RNAs. RNA 18:783–794

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Zhang Y, Heidrich N, Ampattu BJ, Gunderson CW, Seifert HS, Schoen C, Vogel J, Sontheimer EJ (2013) Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol Cell 50:488–503

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Dugar G, Herbig A, Forstner KU, Heidrich N, Reinhardt R, Nieselt K, Sharma CM (2013) High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni Isolates. PLoS Genet 9:e1003495

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Hale CR, Majumdar S, Elmore J, Pfister N, Compton M, Olson S, Resch AM, Glover CV 3rd, Graveley BR, Terns RM, Terns MP (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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP (2009) RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139:945–956

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9:467–477

    Article  CAS  PubMed  Google Scholar 

  30. Richter H, Zoephel J, Schermuly J, Maticzka D, Backofen R, Randau L (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 

  31. Zhang J, Rouillon C, Kerou M, Reeks J, Brugger K, Graham S, Reimann J, Cannone G, Liu H, Albers SV, Naismith JH, Spagnolo L, White MF (2012) Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol Cell 45:303–313

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Bernick DL, Cox CL, Dennis PP, Lowe TM (2012) Comparative genomic and transcriptional analyses of CRISPR systems across the genus Pyrobaculum. Front Microbiol 3:251

    PubMed Central  PubMed  Google Scholar 

  33. Richter H, Lange SJ, Backofen R, Randau L (2013) Comparative analysis of Cas6b processing and CRISPR RNA stability. RNA Biol 10:700–707

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Kroger C, Colgan A, Srikumar S, Handler K, Sivasankaran SK, Hammarlof DL, Canals R, Grissom JE, Conway T, Hokamp K, Hinton JC (2013) An infection-relevant transcriptomic compendium for Salmonella enterica Serovar Typhimurium. Cell Host Microbe 14:683–695

    Article  CAS  PubMed  Google Scholar 

  35. Jager D, Sharma CM, Thomsen J, Ehlers C, Vogel J, Schmitz RA (2009) Deep sequencing analysis of the Methanosarcina mazei Go1 transcriptome in response to nitrogen availability. Proc Natl Acad Sci U S A 106:21878–21882

    Article  PubMed Central  PubMed  Google Scholar 

  36. Zhelyazkova P, Sharma CM, Forstner KU, Liere K, Vogel J, Borner T (2012) The primary transcriptome of barley chloroplasts: numerous noncoding RNAs and the dominating role of the plastid-encoded RNA polymerase. Plant Cell 24:123–136

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Albrecht M, Sharma CM, Reinhardt R, Vogel J, Rudel T (2010) Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome. Nucleic Acids Res 38:868–877

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602–607

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10:94–108

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Carte J, Wang R, Li H, Terns RM, Terns MP (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 

  41. Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA (2010) Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329:1355–1358

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul U, Wurm R, Wagner R, Beijer MR, Barendregt A, Zhou K, Snijders AP, Dickman MJ, Doudna JA, Boekema EJ, Heck AJ, van der Oost J, Brouns SJ (2011) Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol 18:529–536

    Article  CAS  PubMed  Google Scholar 

  43. Blomberg P, Wagner EG, Nordstrom K (1990) Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III. EMBO J 9:2331–2340

    PubMed Central  CAS  PubMed  Google Scholar 

  44. Mattatall NR, Sanderson KE (1996) Salmonella typhimurium LT2 possesses three distinct 23S rRNA intervening sequences. J Bacteriol 178:2272–2278

    PubMed Central  CAS  PubMed  Google Scholar 

  45. Nickel L, Weidenbach K, Jager D, Backofen R, Lange SJ, Heidrich N, Schmitz RA (2013) Two CRISPR-Cas systems in Methanosarcina mazei strain Go1 display common processing features despite belonging to different types I and III. RNA Biol 10:779–791

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  46. Scholz I, Lange SJ, Hein S, Hess WR, Backofen R (2013) CRISPR-Cas systems in the cyanobacterium Synechocystis sp. PCC6803 exhibit distinct processing pathways involving at least two Cas6 and a Cmr2 protein. PLoS One 8:e56470

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  47. Aravin AA, Hannon GJ, Brennecke J (2007) The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 318:761–764

    Article  CAS  PubMed  Google Scholar 

  48. Su AA, Tripp V, Randau L (2013) RNA-Seq analyses reveal the order of tRNA processing events and the maturation of C/D box and CRISPR RNAs in the hyperthermophile Methanopyrus kandleri. Nucleic Acids Res 41:6250–6258

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Soutourina OA, Monot M, Boudry P, Saujet L, Pichon C, Sismeiro O, Semenova E, Severinov K, Le Bouguenec C, Coppee JY, Dupuy B, Martin-Verstraete I (2013) Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile. PLoS Genet 9:e1003493

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  50. Berezikov E, Thuemmler F, van Laake LW, Kondova I, Bontrop R, Cuppen E, Plasterk RH (2006) Diversity of microRNAs in human and chimpanzee brain. Nat Genet 38:1375–1377

    Article  CAS  PubMed  Google Scholar 

  51. Chylinski K, Le Rhun A, Charpentier E (2013) The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol 10:726–737

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  52. Fonfara I, Le Rhun A, Chylinski K, Makarova KS, Lecrivain AL, Bzdrenga J, Koonin EV, Charpentier E (2014) Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res 42:2577–2590

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  53. Konig J, Zarnack K, Luscombe NM, Ule J (2012) Protein-RNA interactions: new genomic technologies and perspectives. Nat Rev Genet 13:77–83

    Article  PubMed  Google Scholar 

  54. Chao Y, Papenfort K, Reinhardt R, Sharma CM, Vogel J (2012) An atlas of Hfq-bound transcripts reveals 3′ UTRs as a genomic reservoir of regulatory small RNAs. EMBO J 31:4005–4019

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  55. Brouns SJJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJH, Snijders APL, Dickman MJ, Makarova KS, Koonin EV, van der Oost J (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964

    Article  CAS  PubMed  Google Scholar 

  56. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

    Article  CAS  PubMed  Google Scholar 

  57. Sampson TR, Saroj SD, Llewellyn AC, Tzeng YL, Weiss DS (2013) A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature 497:254–257

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  58. Heidrich N, Vogel J (2013) CRISPRs extending their reach: prokaryotic RNAi protein Cas9 recruited for gene regulation. EMBO J 32:1802–1804

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  59. Maier LK, Lange SJ, Stoll B, Haas KA, Fischer S, Fischer E, Duchardt-Ferner E, Wohnert J, Backofen R, Marchfelder A (2013) Essential requirements for the detection and degradation of invaders by the Haloferax volcanii CRISPR/Cas system I-B. RNA Biol 10:865–874

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  60. Randau L (2012) RNA processing in the minimal organism Nanoarchaeum equitans. Genome Biol 13:R63

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  61. Elmore JR, Yokooji Y, Sato T, Olson S, Glover CV 3rd, Graveley BR, Atomi H, Terns RM, Terns MP (2013) Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis. RNA Biol 10:828–840

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgements

CRISPR work in the Vogel lab is funded by DFG Grant Vo875/7-1 and the Bavarian BioSysNet program. Work in the Sharma lab is supported by the ZINF Young Investigator program at the Research Center for Infectious Diseases (ZINF) in Würzburg, Germany, the Bavarian BioSysNet program, DFG Grant Sh580/1-1 and the Daimler and Benz foundation. GD is supported by the Graduate School for Life Sciences (GSLS) Würzburg.

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Authors and Affiliations

  1. Institute for Molecular Infection Biology (IMIB), University of Würzburg, Josef-Schneider-Straße 2/Bau D15, 97080, Würzburg, Germany

    Nadja Heidrich & Jörg Vogel

  2. Research Centre for Infectious Diseases (ZINF), University of Würzburg, Josef-Schneider-Straße 2/Bau D15, 97080, Würzburg, Germany

    Gaurav Dugar & Cynthia M. Sharma

Authors
  1. Nadja Heidrich
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  2. Gaurav Dugar
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  3. Jörg Vogel
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  4. Cynthia M. Sharma
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Corresponding author

Correspondence to Cynthia M. Sharma .

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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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Heidrich, N., Dugar, G., Vogel, J., Sharma, C.M. (2015). Investigating CRISPR RNA Biogenesis and Function Using RNA-seq. 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_1

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  • DOI: https://doi.org/10.1007/978-1-4939-2687-9_1

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2686-2

  • Online ISBN: 978-1-4939-2687-9

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