CRISPR pp 195-222 | Cite as

Procedures for Generating CRISPR Mutants with Novel Spacers Acquired from Viruses or Plasmids

  • Marie-Ève Dupuis
  • Rodolphe Barrangou
  • Sylvain Moineau
Part of the Methods in Molecular Biology book series (MIMB, volume 1311)


CRISPR-Cas systems provide immunity in bacteria and archaea against nucleic acids in the form of viral genomes and plasmids, and influence their coevolution. The first main step of CRISPR-Cas activity is the immune adaptation through spacer(s) acquisition into an active CRISPR locus. This step is also mandatory for the final stage of CRISPR-Cas activity, namely interference. This chapter describes general procedures for studying the CRISPR adaptation step, accomplished by producing bacteriophage-insensitive mutants (BIMs) or plasmid-interfering mutants (PIMs) using various spacer acquisition analyses and experiments. Since each bacterial or archaeal species (and even strain) needs specific conditions to optimize the acquisition process, the protocols described below should be thought of as general guidelines and may not be applicable universally, without modification.

Because Streptococcus thermophilus was used as the model system in the first published study on novel spacer acquisition and in many studies ever since, the protocols in this chapter describe specific conditions, media, and buffers that have been used with this microorganism. Details for other species will be given when possible, but readers should first evaluate the best growth and storage conditions for each bacterium—foreign element pair (named the procedure settings) and bear in mind the specificity and variability of CRISPR-Cas types and subtypes. Also, we suggest to be mindful of the fact that some CRISPR-Cas systems are not “naturally” active in terms of the ability to acquire novel CRISPR spacers, and that some systems may require specific conditions to induce the CRISPR-Cas activity for spacer acquisition.

Key words

Acquisition Adaptation Adaptive immunity Bacteriophage insensitive mutants (BIMs) CRISPR Plasmid interfering mutants (PIMs) Protospacer Protospacer adjacent motif (PAM) Spacer Cas 


  1. 1.
    Grissa I, Vergnaud G, Pourcel C (2007) CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:W52–W57CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169:5429–5433PubMedCentralPubMedGoogle Scholar
  3. 3.
    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 315:1709–1712CrossRefPubMedGoogle Scholar
  4. 4.
    Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71CrossRefPubMedGoogle Scholar
  5. 5.
    Makarova KS, Wolf YI, Koonin EV (2013) Comparative genomics of defense systems in archaea and bacteria. Nucleic Acids Res 41:4360–4377CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Marchfelder A (2013) Special focus CRISPR-Cas. RNA Biol 10:655–658CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Koonin EV, Makarova KS (2013) CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes. RNA Biol 10:679–686CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu Rev Biochem 82:237–266CrossRefPubMedGoogle Scholar
  9. 9.
    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
  10. 10.
    Lange SJ, Alkhnbashi OS, Rose D, Will S, Backofen R (2013) CRISPRmap: an automated classification of repeat conservation in prokaryotic adaptive immune systems. Nucleic Acids Res 41:8034–8044CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Yin S, Jensen MA, Bai J, Debroy C, Barrangou R, Dudley EG (2013) Evolutionary divergence of Shiga toxin-producing Escherichia coli is reflected in CRISPR spacer composition. Appl Environ Microbiol 79:5710–5720CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Held NL, Herrera A, Whitaker RJ (2013) Reassortment of CRISPR repeat-spacer loci in Sulfolobus islandicus. Environ Microbiol 15:3065–3076Google Scholar
  13. 13.
    Shariat N, Kirchner MK, Sandt CH, Trees E, Barrangou R, Dudley EG (2013) Subtyping of Salmonella enterica serovar Newport outbreak isolates by CRISPR-MVLST and determination of the relationship between CRISPR-MVLST and PFGE results. J Clin Microbiol 51:2328–2336CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Cai F, Axen SD, Kerfeld CA (2013) Evidence for the widespread distribution of CRISPR-Cas system in the Phylum Cyanobacteria. RNA Biol 10:687–693CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Emerson JB, Andrade K, Thomas BC, Norman A, Allen EE, Heidelberg KB, Banfield JF (2013) Virus-host and CRISPR dynamics in archaea-dominated hypersaline Lake Tyrrell, Victoria, Australia. Archaea 2013:370871CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Minot S, Bryson A, Chehoud C, Wu GD, Lewis JD, Bushman FD (2013) Rapid evolution of the human gut virome. Proc Natl Acad Sci U S A 110:12450–12455CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Zhang Q, Rho M, Tang H, Doak TG, Ye Y (2013) CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes. Genome Biol 14:R40CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C, Boyaval P, Romero DA, Horvath P, Moineau S (2008) Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 190:1390–1400CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Savitskaya E, Semenova E, Dedkov V, Metlitskaya A, Severinov K (2013) High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli. RNA Biol 10:716–725CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Chylinskim K, Le Rhunm A, Charpentier E (2013) The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol 10:726–737CrossRefGoogle Scholar
  21. 21.
    Magadán AH, Dupuis MÈ, Villion M, Moineau S (2012) Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system. PLoS One 7:e40913CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Biswas A, Gagnon JN, Brouns SJ, Fineran PC, Brown CM (2013) CRISPRTarget: bioinformatic prediction and analysis of crRNA targets. RNA Biol 10:817–827CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Shah SA, Erdmann S, Mojica FJ, Garrett RA (2013) Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol 10:891–899CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Peng W, Li H, Hallstrøm S, Peng N, Liang YX, She Q (2013) Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus. RNA Biol 10:738–748CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Almendros C, Guzmán NM, Díez-Villaseñor C, García-Martínez J, Mojica FJ (2012) Target motifs affecting natural immunity by a constitutive CRISPR-Cas system in Escherichia coli. PLoS One 8:e50797CrossRefGoogle Scholar
  26. 26.
    Sun CL, Barrangou R, Thomas BC, Horvath P, Fremaux C, Banfield JF (2013) Phage mutations in response to CRISPR diversification in a bacterial population. Environ Microbiol 15:463–470CrossRefPubMedGoogle Scholar
  27. 27.
    Fischer S, Maier LK, Stoll B, Brendel J, Fischer E, Pfeiffer F, Dyall-Smith M, Marchfelder A (2012) An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA. J Biol Chem 287:33351–33363CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Swarts DC, Mosterd C, van Passel MW, Brouns SJ (2012) CRISPR interference directs strand specific spacer acquisition. PLoS One 7:e35888CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    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
  30. 30.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefPubMedCentralPubMedGoogle Scholar
  31. 31.
    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826CrossRefPubMedCentralPubMedGoogle Scholar
  32. 32.
    Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Cho SW, Kim S, Kim JM, Kim JS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232CrossRefPubMedGoogle Scholar
  34. 34.
    Horvath P, Barrangou R (2013) RNA-guided genome editing à la carte. Cell Res 23:733–734CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918CrossRefPubMedCentralPubMedGoogle Scholar
  36. 36.
    Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA (2013) Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods 10:741–743CrossRefPubMedGoogle Scholar
  37. 37.
    Horvath P, Romero DA, Coûté-Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P, Fremaux C, Barrangou R (2008) Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J Bacteriol 190:1401–1412CrossRefPubMedCentralPubMedGoogle 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.
    Levin BR, Moineau S, Bushman M, Barrangou R (2013) The population and evolutionary dynamics of phage and bacteria with CRISPR-mediated immunity. PLoS One Genet 9:e1003312CrossRefGoogle Scholar
  40. 40.
    Lillehaug D (1997) An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages. J Appl Microbiol 83:85–90CrossRefPubMedGoogle Scholar
  41. 41.
    Paez-Espino D, Morovic W, Sun CL, Thomas BC, Ueda K, Stahl B, Barrangou R, Banfield JF (2013) Strong bias in the bacterial CRISPR elements that confer immunity to phage. Nat Commun 4:1430CrossRefPubMedGoogle Scholar
  42. 42.
    Mills S, Griffin C, Coffey A, Meijer WC, Hafkamp B, Ross RP (2010) CRISPR analysis of bacteriophage-insensitive mutants (BIMs) of industrial Streptococcus thermophilus—implications for starter design. J Appl Microbiol 108:945–955CrossRefPubMedGoogle Scholar
  43. 43.
    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 3:945CrossRefPubMedGoogle Scholar
  44. 44.
    Yosef I, Goren MG, Qimron U (2012) Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nat Acids Res 40:5569–5576CrossRefGoogle Scholar
  45. 45.
    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 77:1367–1379CrossRefPubMedCentralPubMedGoogle Scholar
  46. 46.
    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 75:1495–1512CrossRefPubMedGoogle Scholar
  47. 47.
    Medina-Aparicio L, Rebollar-Flores JE, Gallego-Hernández AL, Vázquez A, Olvera L, Gutiérrez-Ríos RM, Calva E, Hernández-Lucas I (2011) The CRISPR/Cas immune system is an operon regulated by LeuO, H-NS, and leucine-responsive regulatory protein in Salmonella enterica serovar Typhi. J Bacteriol 193:2396–2407CrossRefPubMedCentralPubMedGoogle Scholar
  48. 48.
    Dupuis MÈ, Villion M, Magadán AH, Moineau S (2013) CRISPR-Cas and restriction-modification systems are compatible and increase phage resistance. Nat Commun 4:2087CrossRefPubMedGoogle Scholar
  49. 49.
    Garneau J (2009) Caractérisation du système CRISPR-cas chez Streptococcus thermophilus. Master thesis, University Laval, Quebec, 109 p.
  50. 50.
    Dupuis MÈ (2011) Caractérisation du mode d’action du système CRISPR1/Cas de Streptococcus thermophilus. Master thesis, University Laval, Quebec, 113 p.
  51. 51.
    van der Ploeg JR (2009) Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages. Microbiology 155:1966–1976CrossRefPubMedGoogle Scholar
  52. 52.
    Cady KC, Bondy-Denomy J, Heussler GE, Davidson AR, O’Toole GA (2012) The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages. J Bacteriol 194(5728):5738Google Scholar
  53. 53.
    Erdmann S, Garrett RA (2012) Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms. Mol Microbiol 85:1044–1056CrossRefPubMedCentralPubMedGoogle Scholar
  54. 54.
    Lopez-Sanchez MJ, Sauvage E, Da Cunha V, Clermont D, Ratsima Hariniaina E, Gonzalez-Zorn B, Poyart C, Rosinski-Chupin I, Glaser P (2013) The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome. Mol Microbiol 85:1057–1071CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Marie-Ève Dupuis
    • 1
    • 2
  • Rodolphe Barrangou
    • 3
  • Sylvain Moineau
    • 1
    • 2
  1. 1.Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de GénieUniversité LavalQuebec CityCanada
  2. 2.Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Félix d’Hérelle Reference Center for Bacterial VirusesUniversité LavalQuebec CityCanada
  3. 3.Food, Bioprocessing and Nutrition Sciences DepartmentNorth Carolina State UniversityRaleighUSA

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