CRISPR pp 233-250 | Cite as

Using the CRISPR-Cas System to Positively Select Mutants in Genes Essential for Its Function

  • Ido Yosef
  • Moran G. Goren
  • Rotem Edgar
  • Udi Qimron
Part of the Methods in Molecular Biology book series (MIMB, volume 1311)

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) comprise a prokaryotic adaptive defense system against foreign nucleic acids. This defense is mediated by Cas proteins, which are guided by sequences flanked by the repeats, called spacers, to target nucleic acids. Spacers designed against the prokaryotic self chromosome are lethal to the prokaryotic cell. This self-killing of the bacterium by its own CRISPR-Cas system can be used to positively select genes that participate in this killing, as their absence will result in viable cells. Here we describe a positive selection assay that uses this feature to identify E. coli mutants encoding an inactive CRISPR-Cas system. The procedure includes establishment of an assay that detects this self-killing, generation of transposon insertion mutants in random genes, and selection of viable mutants, suspected as required for this lethal activity. This procedure enabled us to identify a novel gene, htpG, that is required for the activity of the CRISPR-Cas system. The procedures described here can be adjusted to various organisms to identify genes required for their CRISPR-Cas activity.

Key words

Defense mechanism Phage-host interaction Non-cas genes Autoimmunity Positive selection 

Notes

Acknowledgements

This research was supported by the Israel Science Foundation grant 611/10 to U.Q., and the Marie Curie International Reintegration Grant PIRG-2010-266717 to R.E.

References

  1. 1.
    Marraffini LA, Sontheimer EJ (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322(5909):1843–1845. doi: 10.1126/science.1165771 CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    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(5819):1709–1712. doi: 10.1126/science.1138140 CrossRefPubMedGoogle Scholar
  3. 3.
    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(5891):960–964. doi: 10.1126/science.1159689 CrossRefPubMedGoogle Scholar
  4. 4.
    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(5):945–956. doi: 10.1016/j.cell.2009.07.040 CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Goren M, Yosef I, Edgar R, Qimron U (2012) The bacterial CRISPR/Cas system as analog of the mammalian adaptive immune system. RNA Biol 9(5):549–554. doi: 10.4161/rna.20177 CrossRefPubMedGoogle Scholar
  6. 6.
    Abedon ST (2012) Bacterial ‘immunity’ against bacteriophages. Bacteriophage 2(1):50–54. doi: 10.4161/bact.18609 CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Bhaya D, Davison M, Barrangou R (2011) CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 45:273–297. doi: 10.1146/annurev-genet-110410-132430 CrossRefPubMedGoogle Scholar
  8. 8.
    Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482(7385):331–338. doi: 10.1038/nature10886 CrossRefPubMedGoogle 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(6):467–477. doi: 10.1038/nrmicro2577 CrossRefPubMedGoogle Scholar
  10. 10.
    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(6):1367–1379. doi: 10.1111/j.1365-2958.2010.07265.x CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    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. doi: 10.1093/nar/gks216 CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Deveau H, Garneau JE, Moineau S (2010) CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol 64:475–493. doi: 10.1146/annurev.micro.112408.134123 CrossRefPubMedGoogle Scholar
  13. 13.
    Sorek R, Kunin V, Hugenholtz P (2008) CRISPR—a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol 6(3):181–186. doi: 10.1038/nrmicro1793 CrossRefPubMedGoogle Scholar
  14. 14.
    Marraffini LA, Sontheimer EJ (2010) CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 11(3):181–190. doi: 10.1038/nrg2749 CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O’Connor-Giles KM (2013) Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194(4):1029–1035. doi: 10.1534/genetics.113.152710 CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Gaj T, Gersbach CA, Barbas CF III (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31(7):397–405. doi: 10.1016/j.tibtech.2013.04.004 CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    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(4):910–918. doi: 10.1016/j.cell.2013.04.025 CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Blackburn PR, Campbell JM, Clark KJ, Ekker SC (2013) The CRISPR system—keeping zebrafish gene targeting fresh. Zebrafish 10(1):116–118.  doi: 10.1089/zeb.2013.9999 CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7):4336–4343. doi: 10.1093/nar/gkt135 CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Ramalingam S, Annaluru N, Chandrasegaran S (2013) A CRISPR way to engineer the human genome. Genome Biol 14(2):107. doi: 10.1186/gb-2013-14-2-107 CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31(3):233–239. doi: 10.1038/nbt.2508 CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    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(6121):819–823.  doi: 10.1126/science.1231143 CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Edgar R, Qimron U (2010) The Escherichia coli CRISPR system protects from lambda lysogenization, lysogens, and prophage induction. J Bacteriol 192(23):6291–6294. doi: 10.1128/JB.00644-10 CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Yosef I, Goren MG, Kiro R, Edgar R, Qimron U (2011) High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. Proc Natl Acad Sci U S A 108(50):20136–20141. doi: 10.1073/pnas.1113519108 CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Vercoe RB, Chang JT, Dy RL, Taylor C, Gristwood T, Clulow JS, Richter C, Przybilski R, Pitman AR, Fineran PC (2013) Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet 9(4):e1003454. doi: 10.1371/journal.pgen.1003454 CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, Mori H (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 12(5):291–299. doi: 10.1093/dnares/dsi012 CrossRefPubMedGoogle Scholar
  27. 27.
    Wilson K (1994) Preparation of genomic DNA from bacteria, p. 2.4. 1-2.4. 5. InIn FA Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, and K. Struhl. Current protocols in molecular biology John Wiley & Sons, Inc, New York, NYGoogle Scholar
  28. 28.
    Larsen RA, Wilson MM, Guss AM, Metcalf WW (2002) Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol 178(3):193–201. doi: 10.1007/s00203-002-0442-2 CrossRefPubMedGoogle Scholar
  29. 29.
    Walker CB, Stolyar S, Chivian D, Pinel N, Gabster JA, Dehal PS, He Z, Yang ZK, Yen HC, Zhou J, Wall JD, Hazen TC, Arkin AP, Stahl DA (2009) Contribution of mobile genetic elements to Desulfovibrio vulgaris genome plasticity. Environ Microbiol 11(9):2244–2252. doi: 10.1111/j.1462-2920.2009.01946.x CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Ido Yosef
    • 1
  • Moran G. Goren
    • 1
  • Rotem Edgar
    • 1
  • Udi Qimron
    • 1
  1. 1.Department of Clinical Microbiology and Immunology, Sackler Faculty of MedicineTel Aviv UniversityTel AvivIsrael

Personalised recommendations