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Horizontal Gene Transfer pp 235–249Cite as

Methods for the Analysis and Characterization of Defense Mechanisms Against Horizontal Gene Transfer: CRISPR Systems

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2075))

Abstract

CRISPR-Cas systems provide RNA-guided adaptive immunity to the majority of archaea and many bacteria. They are able to capture pieces of invading genetic elements in the form of novel spacers in an array of repeats. These elements can then be used as a memory to destroy incoming DNA through the action of RNA-guided nucleases. This chapter describes general procedures to determine the ability of CRISPR-Cas systems to capture novel sequences and to use them to block phages and horizontal gene transfer. All protocols are performed in Staphylococcus aureus using Type II-A CRISPR-Cas systems. Nonetheless, the protocols provided can be adapted to work with other bacteria and other types of CRISPR-Cas systems.

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References

  1. Makarova KS, Wolf YI, Alkhnbashi OS et al (2015) An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol 13:722–736. https://doi.org/10.1038/nrmicro3569

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Bolotin A, Quinquis B, Sorokin A, Dusko Ehrlich S (2005) Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151:2551–2561. https://doi.org/10.1099/mic.0.28048-0

    Article  CAS  PubMed  Google Scholar 

  3. Mojica FJM, Díez-Villaseñor C, García-Martínez J, Soria E (2005) Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 60:174–182. https://doi.org/10.1007/s00239-004-0046-3

    Article  CAS  PubMed  Google Scholar 

  4. Pourcel C, Salvignol G, Vergnaud G (2005) CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151:653–663. https://doi.org/10.1099/mic.0.27437-0

    Article  CAS  PubMed  Google Scholar 

  5. Barrangou R, Fremaux C, Deveau H et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712. https://doi.org/10.1126/science.1138140

    Article  CAS  PubMed  Google Scholar 

  6. Marraffini LA, Sontheimer EJ (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322:1843–1845. https://doi.org/10.1126/science.1165771

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Garneau JE, Dupuis MÈ, Villion M et al (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71. https://doi.org/10.1038/nature09523

    Article  CAS  PubMed  Google Scholar 

  8. Bikard D, Hatoum-Aslan A, Mucida D, Marraffini LA (2012) CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. Cell Host Microbe 12:177–186. https://doi.org/10.1016/j.chom.2012.06.003

    Article  CAS  PubMed  Google Scholar 

  9. Grissa I, Vergnaud G, Pourcel C (2007) CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:W52–W57. https://doi.org/10.1093/nar/gkm360

    Article  PubMed  PubMed Central  Google Scholar 

  10. Biswas A, Staals RHJ, Morales SE et al (2016) CRISPRDetect: a flexible algorithm to define CRISPR arrays. BMC Genomics 17:356. https://doi.org/10.1186/s12864-016-2627-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Koonin EV, Makarova KS, Zhang F (2017) Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol 37:67–78. https://doi.org/10.1016/j.mib.2017.05.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Shmakov S, Smargon A, Scott D et al (2017) Diversity and evolution of class 2 CRISPR–Cas systems. Nat Rev Microbiol 15:169–182. https://doi.org/10.1038/nrmicro.2016.184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sinkunas T, Gasiunas G, Fremaux C et al (2011) Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J 30:1335–1342. https://doi.org/10.1038/emboj.2011.41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gong B, Shin M, Sun J et al (2014) Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3. Proc Natl Acad Sci 111:16359–16364. https://doi.org/10.1073/pnas.1410806111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Huo Y, Nam KH, Ding F et al (2014) Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation. Nat Struct Mol Biol 21:771–777. https://doi.org/10.1038/nsmb.2875

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–822. https://doi.org/10.1126/science.1225829

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zetsche B, Gootenberg JS, Abudayyeh OO et al (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771. https://doi.org/10.1016/j.cell.2015.09.038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shmakov S, Abudayyeh OO, Makarova KS et al (2015) Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol Cell 60:385–397. https://doi.org/10.1016/j.molcel.2015.10.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Abudayyeh OO, Gootenberg JS, Konermann S et al (2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353:aaf5573. https://doi.org/10.1126/science.aaf5573

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yan WX, Hunnewell P, Alfonse LE et al (2019) Functionally diverse type V CRISPR-Cas systems. Science 363(6422):88–91. https://doi.org/10.1126/SCIENCE.AAV7271

    Article  CAS  PubMed  Google Scholar 

  21. Modell JW, Jiang W, Marraffini LA (2017) CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity. Nature 544:101–104. https://doi.org/10.1038/nature21719

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wiedenheft B, Zhou K, Jinek M et al (2009) Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. Structure 17:904–912. https://doi.org/10.1016/j.str.2009.03.019

    Article  CAS  PubMed  Google Scholar 

  23. Beloglazova N, Brown G, Zimmerman MD et al (2008) A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats. J Biol Chem 283:20361–20371. https://doi.org/10.1074/jbc.M803225200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yosef I, Goren MG, Qimron U (2012) Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res 40:5569–5576. https://doi.org/10.1093/nar/gks216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Arslan Z, Hermanns V, Wurm R et al (2014) Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system. Nucleic Acids Res 42:7884–7893. https://doi.org/10.1093/nar/gku510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Nuñez JK, Kranzusch PJ, Noeske J et al (2014) Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity. Nat Struct Mol Biol 21:528–534

    Article  PubMed  PubMed Central  Google Scholar 

  27. Heler R, Samai P, Modell JW et al (2015) Cas9 specifies functional viral targets during CRISPR-Cas adaptation. Nature 519:199–202

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Swarts DC, Mosterd C, van Passel MWJ, Brouns SJJ (2012) CRISPR interference directs strand specific spacer acquisition. PLoS One 7:e35888. https://doi.org/10.1371/journal.pone.0035888

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Leenay RT, Maksimchuk KR, Slotkowski RA et al (2015) Identifying and visualizing functional PAM diversity across CRISPR-Cas systems. Mol Cell 62:1–11. https://doi.org/10.1016/j.molcel.2016.02.031

    Article  CAS  Google Scholar 

  30. Karvelis T, Gasiunas G, Siksnys V (2017) Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: a brief overview. Methods 121–122:3–8. https://doi.org/10.1016/j.ymeth.2017.03.006

    Article  CAS  PubMed  Google Scholar 

  31. Datsenko KA, Pougach K, Tikhonov A et al (2012) Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat Commun 3:945–947. https://doi.org/10.1038/ncomms1937

    Article  CAS  PubMed  Google Scholar 

  32. Fineran PC, Charpentier E (2012) Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new information. Virology 434:202–209. https://doi.org/10.1016/j.virol.2012.10.003

    Article  CAS  PubMed  Google Scholar 

  33. Semenova E, Savitskaya E, Musharova O et al (2016) Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex. Proc Natl Acad Sci 113:7626–7631. https://doi.org/10.1073/pnas.1602639113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Brouns SJJ, Jore MM, Lundgren M et al (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964. https://doi.org/10.1126/science.1159689

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Deltcheva E, Chylinski K, Sharma CM et al (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602–607. https://doi.org/10.1038/nature09886

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Haurwitz RE, Jinek M, Wiedenheft B et al (2010) Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329:1355–1358. https://doi.org/10.1126/science.1192272

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 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. https://doi.org/10.1101/gad.1742908

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Sternberg SH, Redding S, Jinek M et al (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507:62–67. https://doi.org/10.1038/nature13011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Marraffini LA (2015) CRISPR-Cas immunity in prokaryotes. Nature 526:55–61. https://doi.org/10.1038/nature15386

    Article  CAS  PubMed  Google Scholar 

  40. Semenova E, Jore MM, Datsenko KA 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. https://doi.org/10.1073/pnas.1104144108

    Article  PubMed  PubMed Central  Google Scholar 

  41. Wiedenheft B, van Duijn E, Bultema JB et al (2011) RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci 108:10092–10097. https://doi.org/10.1073/pnas.1102716108

    Article  PubMed  PubMed Central  Google Scholar 

  42. Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci 109:E2579–E2586. https://doi.org/10.1073/pnas.1208507109

    Article  PubMed  PubMed Central  Google Scholar 

  43. Samai P, Pyenson N, Jiang W et al (2015) Co-transcriptional DNA and RNA cleavage during type III CRISPR-cas immunity. Cell 161:1164–1174. https://doi.org/10.1016/j.cell.2015.04.027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Jiang W, Samai P, Marraffini LA (2016) Degradation of phage transcripts by CRISPR-associated RNases enables type III CRISPR-Cas immunity. Cell 164:710–721. https://doi.org/10.1016/j.cell.2015.12.053

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Marraffini LA, Sontheimer EJ (2010) Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463:568–571. https://doi.org/10.1038/nature08703

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Dupuis M-È, Barrangou R, Moineau S (2015) Procedures for generating CRISPR mutants with novel spacers acquired from viruses or plasmids. In: Lundgren M, Charpentier E, Fineran PC (eds) CRISPR: methods and protocols. Springer New York, New York, NY, pp 195–222

    Chapter  Google Scholar 

  47. Almendros C, Mojica FJM (2015) Exploring CRISPR interference by transformation with plasmid mixtures: identification of target interference motifs in Escherichia coli. In: Lundgren M, Charpentier E, Fineran PC (eds) CRISPR: methods and protocols. Springer New York, New York, NY, pp 161–170

    Chapter  Google Scholar 

  48. Heidrich N, Dugar G, Vogel J, Sharma CM (2015) Investigating CRISPR RNA biogenesis and function using RNA-seq. In: Lundgren M, Charpentier E, Fineran PC (eds) CRISPR: methods and protocols. Springer New York, New York, NY, pp 1–21

    Google Scholar 

  49. Waghmare SP, Nwokeoji AO, Dickman MJ (2015) Analysis of crRNA using liquid chromatography electrospray ionization mass spectrometry (LC ESI MS). In: Lundgren M, Charpentier E, Fineran PC (eds) CRISPR: methods and protocols. Springer New York, New York, NY, pp 133–145

    Chapter  Google Scholar 

  50. Garside EL, MacMillan AM (2015) Analysis of CRISPR pre-crRNA cleavage. In: Lundgren M, Charpentier E, Fineran PC (eds) CRISPR: methods and protocols. Springer New York, New York, NY, pp 35–46

    Chapter  Google Scholar 

  51. Leenay RT, Beisel CL (2017) Deciphering, communicating, and engineering the CRISPR PAM. J Mol Biol 429:177–191. https://doi.org/10.1016/j.jmb.2016.11.024

    Article  CAS  PubMed  Google Scholar 

  52. Edgar R, Qimron U (2010) The Escherichia coli CRISPR system protects from λ lysogenization, lysogens, and prophage induction. J Bacteriol 192:6291–6294. https://doi.org/10.1128/JB.00644-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Labrie SJ, Samson JE, Moineau S (2010) Bacteriophage resistance mechanisms. Nat Rev Microbiol 8:317–327. https://doi.org/10.1038/nrmicro2315

    Article  CAS  PubMed  Google Scholar 

  54. Goldberg GW, Jiang W, Bikard D, Marraffini LA (2014) Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting. Nature 514:633–637. https://doi.org/10.1038/nature13637

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Bikard D, Euler CW, Jiang W et al (2014) Exploiting CRISPR-cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol 32:1146–1150. https://doi.org/10.1038/nbt.3043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Bae T, Baba T, Hiramatsu K, Schneewind O (2006) Prophages of Staphylococcus aureus Newman and their contribution to virulence. Mol Microbiol 62:1035–1047. https://doi.org/10.1111/j.1365-2958.2006.05441.x

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Synthetic Biology Group, Microbiology Department, Institut Pasteur, Paris, France

    Alicia Calvo-Villamañán, Aude Bernheim & David Bikard

  2. École Doctorale FIRE—Programme Bettencourt, Centre de Recherches Interdisciplinaires, Paris, France

    Alicia Calvo-Villamañán & Aude Bernheim

  3. Microbial Evolutionary Genomics, Department of Genomes and Genetics, Institut Pasteur, Paris, France

    Aude Bernheim

  4. AgroParisTech, Paris, France

    Aude Bernheim

Authors
  1. Alicia Calvo-Villamañán
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  2. Aude Bernheim
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  3. David Bikard
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Corresponding author

Correspondence to David Bikard .

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Editors and Affiliations

  1. Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria-CSIC, Santander, Cantabria, Spain

    Fernando de la Cruz

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Calvo-Villamañán, A., Bernheim, A., Bikard, D. (2020). Methods for the Analysis and Characterization of Defense Mechanisms Against Horizontal Gene Transfer: CRISPR Systems. In: de la Cruz, F. (eds) Horizontal Gene Transfer. Methods in Molecular Biology, vol 2075. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9877-7_17

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  • DOI: https://doi.org/10.1007/978-1-4939-9877-7_17

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  • Print ISBN: 978-1-4939-9876-0

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