Advertisement

CRISPR pp 1-21 | Cite as

Investigating CRISPR RNA Biogenesis and Function Using RNA-seq

  • Nadja Heidrich
  • Gaurav Dugar
  • Jörg Vogel
  • Cynthia M. Sharma
Part of the Methods in Molecular Biology book series (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.

Key words

RNA-sequencing (RNA-seq) Deep sequencing cDNA library Terminator exonuclease Differential RNA-seq (dRNA-seq) PNK Small RNA identification CRISPR-Cas tracrRNA crRNA 

Notes

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.

References

  1. 1.
    Mutz KO, Heilkenbrinker A, Lonne M, Walter JG, Stahl F (2013) Transcriptome analysis using next-generation sequencing. Curr Opin Biotechnol 24:22–30CrossRefPubMedGoogle Scholar
  2. 2.
    Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Croucher NJ, Thomson NR (2010) Studying bacterial transcriptomes using RNA-seq. Curr Opin Microbiol 13:619–624CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    van Vliet AH (2010) Next generation sequencing of microbial transcriptomes: challenges and opportunities. FEMS Microbiol Lett 302:1–7CrossRefPubMedGoogle Scholar
  5. 5.
    Sorek R, Cossart P (2010) Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity. Nat Rev Genet 11:9–16CrossRefPubMedGoogle Scholar
  6. 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:e1000163CrossRefPubMedCentralPubMedGoogle Scholar
  7. 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 464:250–255CrossRefPubMedGoogle Scholar
  8. 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 25:584–592CrossRefPubMedGoogle Scholar
  9. 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:e148CrossRefPubMedCentralPubMedGoogle Scholar
  10. 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 5:613–619CrossRefPubMedGoogle Scholar
  11. 11.
    He Y, Vogelstein B, Velculescu VE, Papadopoulos N, Kinzler KW (2008) The antisense transcriptomes of human cells. Science 322:1855–1857CrossRefPubMedCentralPubMedGoogle Scholar
  12. 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:e123CrossRefPubMedCentralPubMedGoogle Scholar
  13. 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 105:3805–3810CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Raabe CA, Tang TH, Brosius J, Rozhdestvensky TS (2014) Biases in small RNA deep sequencing data. Nucleic Acids Res 42:1414–1426CrossRefPubMedCentralPubMedGoogle Scholar
  15. 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 31:1015–1022CrossRefGoogle Scholar
  16. 16.
    Thompson JA, Radonovich MF, Salzman NP (1979) Characterization of the 5′-terminal structure of simian virus 40 early mRNA’s. J Virol 31:437–446PubMedCentralPubMedGoogle Scholar
  17. 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 11:941–950CrossRefPubMedGoogle Scholar
  18. 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 31:6435–6443CrossRefPubMedCentralPubMedGoogle Scholar
  19. 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 93:7794–7799CrossRefPubMedCentralPubMedGoogle Scholar
  20. 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 20:133–141CrossRefPubMedCentralPubMedGoogle Scholar
  21. 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:e7526CrossRefPubMedCentralPubMedGoogle Scholar
  22. 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 27:1043–1049CrossRefPubMedGoogle Scholar
  23. 23.
    Sharma CM, Vogel J (2014) Differential RNA-seq: the approach behind and the biological insight gained. Curr Opin Microbiol 19:97–105CrossRefPubMedGoogle Scholar
  24. 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 18:783–794CrossRefPubMedCentralPubMedGoogle Scholar
  25. 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 50:488–503CrossRefPubMedCentralPubMedGoogle Scholar
  26. 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:e1003495CrossRefPubMedCentralPubMedGoogle Scholar
  27. 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 45:292–302CrossRefPubMedCentralPubMedGoogle Scholar
  28. 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 139:945–956CrossRefPubMedCentralPubMedGoogle Scholar
  29. 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 9:467–477CrossRefPubMedGoogle Scholar
  30. 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 40:9887–9896CrossRefPubMedCentralPubMedGoogle Scholar
  31. 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 45:303–313CrossRefPubMedCentralPubMedGoogle Scholar
  32. 32.
    Bernick DL, Cox CL, Dennis PP, Lowe TM (2012) Comparative genomic and transcriptional analyses of CRISPR systems across the genus Pyrobaculum. Front Microbiol 3:251PubMedCentralPubMedGoogle Scholar
  33. 33.
    Richter H, Lange SJ, Backofen R, Randau L (2013) Comparative analysis of Cas6b processing and CRISPR RNA stability. RNA Biol 10:700–707CrossRefPubMedCentralPubMedGoogle Scholar
  34. 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 14:683–695CrossRefPubMedGoogle Scholar
  35. 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 106:21878–21882CrossRefPubMedCentralPubMedGoogle Scholar
  36. 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 24:123–136CrossRefPubMedCentralPubMedGoogle Scholar
  37. 37.
    Albrecht M, Sharma CM, Reinhardt R, Vogel J, Rudel T (2010) Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome. Nucleic Acids Res 38:868–877CrossRefPubMedCentralPubMedGoogle Scholar
  38. 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 471:602–607CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10:94–108CrossRefPubMedCentralPubMedGoogle Scholar
  40. 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 22:3489–3496CrossRefPubMedCentralPubMedGoogle Scholar
  41. 41.
    Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA (2010) Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329:1355–1358CrossRefPubMedCentralPubMedGoogle Scholar
  42. 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 18:529–536CrossRefPubMedGoogle Scholar
  43. 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 9:2331–2340PubMedCentralPubMedGoogle Scholar
  44. 44.
    Mattatall NR, Sanderson KE (1996) Salmonella typhimurium LT2 possesses three distinct 23S rRNA intervening sequences. J Bacteriol 178:2272–2278PubMedCentralPubMedGoogle Scholar
  45. 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 10:779–791CrossRefPubMedCentralPubMedGoogle Scholar
  46. 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:e56470CrossRefPubMedCentralPubMedGoogle Scholar
  47. 47.
    Aravin AA, Hannon GJ, Brennecke J (2007) The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 318:761–764CrossRefPubMedGoogle Scholar
  48. 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 41:6250–6258CrossRefPubMedCentralPubMedGoogle Scholar
  49. 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:e1003493CrossRefPubMedCentralPubMedGoogle Scholar
  50. 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 38:1375–1377CrossRefPubMedGoogle Scholar
  51. 51.
    Chylinski K, Le Rhun A, Charpentier E (2013) The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol 10:726–737CrossRefPubMedCentralPubMedGoogle Scholar
  52. 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 42:2577–2590CrossRefPubMedCentralPubMedGoogle Scholar
  53. 53.
    Konig J, Zarnack K, Luscombe NM, Ule J (2012) Protein-RNA interactions: new genomic technologies and perspectives. Nat Rev Genet 13:77–83CrossRefPubMedGoogle Scholar
  54. 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 31:4005–4019CrossRefPubMedCentralPubMedGoogle Scholar
  55. 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 321:960–964CrossRefPubMedGoogle Scholar
  56. 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 337:816–821CrossRefPubMedGoogle Scholar
  57. 57.
    Sampson TR, Saroj SD, Llewellyn AC, Tzeng YL, Weiss DS (2013) A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature 497:254–257CrossRefPubMedCentralPubMedGoogle Scholar
  58. 58.
    Heidrich N, Vogel J (2013) CRISPRs extending their reach: prokaryotic RNAi protein Cas9 recruited for gene regulation. EMBO J 32:1802–1804CrossRefPubMedCentralPubMedGoogle Scholar
  59. 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 10:865–874CrossRefPubMedCentralPubMedGoogle Scholar
  60. 60.
    Randau L (2012) RNA processing in the minimal organism Nanoarchaeum equitans. Genome Biol 13:R63CrossRefPubMedCentralPubMedGoogle Scholar
  61. 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 10:828–840CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Nadja Heidrich
    • 1
  • Gaurav Dugar
    • 2
  • Jörg Vogel
    • 1
  • Cynthia M. Sharma
    • 2
  1. 1.Institute for Molecular Infection Biology (IMIB)University of WürzburgWürzburgGermany
  2. 2.Research Centre for Infectious Diseases (ZINF)University of WürzburgWürzburgGermany

Personalised recommendations