Skip to main content
SpringerLink
Log in

crRNA Biogenesis

  • Chapter
  • First Online:
CRISPR-Cas Systems

Abstract

Mature crRNAs are key elements in CRISPR-Cas defense against genome invaders. These short RNAs are composed of unique repeat/spacer sequences that guide the Cas protein(s) to the cognate invading nucleic acids for their destruction. The biogenesis of mature crRNAs involves highly precise processing events. Interestingly, different types of CRISPR-Cas systems have evolved distinct crRNA maturation mechanisms. The CRISPR repeat-spacer array is transcribed as a precursor CRISPR RNA molecule (pre-crRNA) that undergoes one or two maturation steps. In type I CRISPR-Cas systems, pre-crRNA is cleaved within the repeat regions by a specific Cas6-like endoribonuclease that at least in some cases is a subunit of a Cascade complex to yield the mature crRNAs. In type III systems, the standalone endoribonuclease Cas6 processes pre-crRNA by cleavage within the repeats, producing an intermediate molecule that is further trimmed to generate the mature crRNAs. Type II systems have evolved a unique crRNA biogenesis pathway, in which a trans-acting small RNA (encoded by the CRISPR-Cas locus) base pairs with each repeat sequence of the pre-crRNA to form a double-stranded RNA template that is cleaved by the housekeeping endoribonuclease III in the presence of protein Cas9 (Csn1). The generated intermediates are then subjected to further maturation by a yet to be revealed mechanism. In this chapter, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in crRNA biogenesis in the three types of CRISPR-Cas systems.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Baranova N, Nikaido H (2002) The BaeSR two-component regulatory system activates transcription of the yegMNOB (mdtABCD) transporter gene cluster in Escherichia coli and increases its resistance to novobiocin and deoxycholate. J Bacteriol 184:4168–4176

    Article  PubMed  CAS  Google Scholar 

  • Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712

    Article  PubMed  CAS  Google Scholar 

  • Beloglazova N, Petit P, Flick R, Brown G, Savchenko A, Yakunin AF (2011) Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference. EMBO J 30:4616–4627

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Cady KC, O’Toole GA (2011) Non-identity targeting of Yersinia-subtype CRISPR-prophage interaction requires the Csy and Cas3 proteins. J Bacteriol 193:3433–3445

    Article  PubMed  CAS  Google Scholar 

  • Carte J, Pfister NT, Compton MM, Terns RM, Terns MP (2010) Binding and cleavage of CRISPR RNA by Cas6. RNA 16:2181–2188

    Article  PubMed  CAS  Google Scholar 

  • 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  CAS  Google Scholar 

  • Chen L, Brugger K, Skovgaard M, Redder P, She Q, Torarinsson E, Greve B, Awayez M, Zibat A, Klenk HP et al (2005) The genome of Sulfolobus acidocaldarius, a model organism of the crenarchaeota. J Bacteriol 187:4992–4999

    Article  PubMed  CAS  Google Scholar 

  • Datsenko KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E (2012) Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat Commun 3:945

    Article  PubMed  Google Scholar 

  • 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  CAS  Google Scholar 

  • Ebihara A, Yao M, Masui R, Tanaka I, Yokoyama S, Kuramitsu S (2006) Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain. Protein Sci 15:1494–1499

    Article  PubMed  CAS  Google Scholar 

  • Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadan AH, Moineau S (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71

    Article  PubMed  CAS  Google Scholar 

  • Gesner EM, Schellenberg MJ, Garside EL, George MM, Macmillan AM (2011) Recognition and maturation of effector RNAs in a CRISPR interference pathway. Nat Struct Mol Biol 18:688–692

    Article  PubMed  CAS  Google Scholar 

  • Gottesman S (2011) Microbiology: dicing defence in bacteria. Nature 471:588–589

    Article  PubMed  CAS  Google Scholar 

  • Haft DH, Selengut J, Mongodin EF, Nelson KE (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  Google Scholar 

  • Hale C, Kleppe K, Terns RM, Terns MP (2008) Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 14:2572–2579

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • 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  CAS  Google Scholar 

  • Hatoum-Aslan A, Maniv I, Marraffini LA (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

    Article  PubMed  CAS  Google Scholar 

  • Haurwitz RE, Sternberg SH, Doudna JA (2012) Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA. EMBO J 31(12):2824–2832

    Article  PubMed  CAS  Google Scholar 

  • 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  CAS  Google Scholar 

  • Hommais F, Krin E, Laurent-Winter C, Soutourina O, Malpertuy A, Le Caer JP, Danchin A, Bertin P (2001) Large-scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid-associated protein, H–NS. Mol Microbiol 40:20–36

    Article  PubMed  CAS  Google Scholar 

  • Howard JA, Delmas S, Ivancic-Bace I, Bolt EL (2011) Helicase dissociation and annealing of RNA-DNA hybrids by Escherichia coli Cas3 protein. Biochem J 439:85–95

    Article  PubMed  CAS  Google Scholar 

  • 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 Jun 28, (Epub ahead of print)

    Google Scholar 

  • Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul U, Wurm R, Wagner R et al (2011) Structural basis for CRISPR RNA-guided DNA recognition by cascade. Nat Struct Mol Biol 18:529–536

    Article  PubMed  CAS  Google Scholar 

  • 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(4):783–794

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  • Lillestol RK, Redder P, Garrett RA, Brugger K (2006) A putative viral defence mechanism in archaeal cells. Archaea 2:59–72

    Article  PubMed  Google Scholar 

  • Lillestol RK, Shah SA, Brugger K, Redder P, Phan H, Christiansen J, Garrett RA (2009) CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties. Mol Microbiol 72:259–272

    Article  PubMed  Google Scholar 

  • Lintner NG, Kerou M, Brumfield SK, Graham S, Liu H, Naismith JH, Sdano M, Peng N, She Q, Copie V 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

    Article  PubMed  CAS  Google Scholar 

  • Makarova KS, Aravind L, Grishin NV, Rogozin IB, Koonin EV (2002) A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis. Nucleic Acids Res 30:482–496

    Article  PubMed  CAS  Google Scholar 

  • Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV (2006) 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 1:7

    Article  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin A et al (2011b) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9:467–477

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Marraffini LA, Sontheimer EJ (2010) Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463:568–571

    Article  PubMed  CAS  Google Scholar 

  • Mulepati S, Bailey S (2011) Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3). J Biol Chem 286:31896–31903

    Article  PubMed  CAS  Google Scholar 

  • Nam KH, Haitjema C, Liu X, Ding F, Wang H, Delisa MP, Ke A (2012) Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in subtype I-C/Dvulg CRISPR-Cas system. Structure Jul 24, (Epub ahead of print)

    Google Scholar 

  • Oshima T, Ishikawa S, Kurokawa K, Aiba H, Ogasawara N (2006) Escherichia coli histone-like protein H-NS preferentially binds to horizontally acquired DNA in association with RNA polymerase. DNA Res 13:141–153

    Article  PubMed  CAS  Google Scholar 

  • Perez-Rodriguez R, Haitjema C, Huang Q, Nam KH, Bernardis S, Ke A, DeLisa MP (2011) Envelope stress is a trigger of CRISPR RNA-mediated DNA silencing in Escherichia coli. Mol Microbiol 79:584–599

    Article  PubMed  CAS  Google Scholar 

  • Phok K, Moisan A, Rinaldi D, Brucato N, Carpousis AJ, Gaspin C, Clouet-d’Orval B (2011) Identification of CRISPR and riboswitch related RNAs among novel noncoding RNAs of the euryarchaeon Pyrococcus abyssi. BMC Genomics 12:312

    Article  PubMed  CAS  Google Scholar 

  • Plagens A, Tjaden B, Hagemann A, Randau L, Hensel R (2012) Characterization of the CRISPR/Cas subtype I-A system of the hyperthermophilic crenarchaeon Thermoproteus tenax. J Bacteriol 194(10):2491–2500

    Article  PubMed  CAS  Google Scholar 

  • Pougach K, Semenova E, Bogdanova E, Datsenko KA, Djordjevic M, Wanner BL, Severinov K (2010) Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol Microbiol 77:1367–1379

    Article  PubMed  CAS  Google Scholar 

  • Pul U, Wurm R, Arslan Z, Geissen R, Hofmann N, Wagner R (2010) Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H–NS. Mol Microbiol 75:1495–1512

    Article  PubMed  CAS  Google Scholar 

  • Randau L (2012). RNA processing in the minimal organism Nanoarchaeum equitans. Genome Biol 13(7):R63, (Epub ahead of print)

    Google Scholar 

  • Richter H, Zoephel J, Schermuly J, Maticzka D, Backofen R and Randau L (2012). Characterization of CRISPR RNA processing in Clostridium thermocellum and Methanococcus maripaludis. Nucleic Acids Res Aug 8, (Epub ahead of print)

    Google Scholar 

  • Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V (2011) The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res 39:9275–9282

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, Severinov K (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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V (2011) Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J 30:1335–1342

    Article  PubMed  CAS  Google Scholar 

  • Sternberg SH, Haurwitz RE, Doudna JA (2012) Mechanism of substrate selection by a highly specific CRISPR endoribonuclease. RNA 18(4):661–672

    Article  PubMed  CAS  Google Scholar 

  • Swarts DC, Mosterd C, van Passel MW, Brouns SJ (2012) CRISPR interference directs strand specific spacer acquisition. PLoS ONE 7(4):e35888

    Article  PubMed  CAS  Google Scholar 

  • Tang TH, Bachellerie JP, Rozhdestvensky T, Bortolin ML, Huber H, Drungowski M, Elge T, Brosius J, Huttenhofer A (2002) Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc Natl Acad Sci U S A 99:7536–7541

    Article  PubMed  CAS  Google Scholar 

  • Tang TH, Polacek N, Zywicki M, Huber H, Brugger K, Garrett R, Bachellerie JP, Huttenhofer A (2005) Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus. Mol Microbiol 55:469–481

    Article  PubMed  CAS  Google Scholar 

  • van der Oost J, Jore MM, Westra ER, Lundgren M, Brouns SJ (2009) CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem Sci 34:401–407

    Article  PubMed  Google Scholar 

  • Vaucheret H (2005). MicroRNA-dependent trans-acting siRNA production. Sci STKE 2005, pe43

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Westra ER, Pul U, Heidrich N, Jore MM, Lundgren M, Stratmann T, Wurm R, Raine A, Mescher M, Van Heereveld L 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

    Article  PubMed  CAS  Google Scholar 

  • Wiedenheft B, Lander GC, Zhou K, Jore MM, Brouns SJ, van der Oost J, Doudna JA, Nogales E (2011a) Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477:486–489

    Article  PubMed  CAS  Google Scholar 

  • Wiedenheft B, van Duijn E, Bultema JB, Waghmare SP, Zhou K, Barendregt A, Westphal W, Heck AJ, Boekema EJ, Dickman MJ et al (2011b) 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

    Article  PubMed  CAS  Google Scholar 

  • Yosef I, Goren MG, Qimron U (2012) Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res 40(12):5569–5576

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå, Sweden

    Emmanuelle Charpentier

  2. Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands

    John van der Oost

  3. Biomedical Sciences Research Complex, University of St Andrews, St Andrews, Fife, KY16 9ST, UK

    Malcolm F. White

  4. Helmholtz Centre for Infection Research, Braunschweig, Germany

    Emmanuelle Charpentier

  5. Hannover Medical School, Hannover, Germany

    Emmanuelle Charpentier

Authors
  1. Emmanuelle Charpentier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John van der Oost
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Malcolm F. White
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Emmanuelle Charpentier .

Editor information

Editors and Affiliations

  1. DuPont Nutrition and Health, Agriculture Drive 3329, Madison, 53716, Wisconsin, USA

    Rodolphe Barrangou

  2. , Laboratory of Microbiology, University of Wageningen, Dreijenplein 10, Wageningen, 6703 HB, Netherlands

    John van der Oost

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer-Verlag Berlin Heidelberg

About this chapter

Cite this chapter

Charpentier, E., van der Oost, J., White, M.F. (2013). crRNA Biogenesis. In: Barrangou, R., van der Oost, J. (eds) CRISPR-Cas Systems. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-34657-6_5

Download citation

Publish with us

Policies and ethics

Search

Navigation