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Selection and Validation of Spacer Sequences for CRISPR-Cas9 Genome Editing and Transcription Regulation in Bacteria

  • Frédéric Grenier
  • Jean-François Lucier
  • Sébastien RodrigueEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1334)

Abstract

RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems have recently been adapted as sequence-programmable tools for various purposes such as genome editing and transcriptional regulation. A critical aspect of the system is the selection and validation of spacer sequences that allow precise targeting of the guide RNA-Cas9 complex. We describe a procedure involving computational and experimental steps to identify and test potentially interesting spacer sequences in bacterial genomes.

Key words

CRISPR Cas9 gRNA Genome editing Transcription Repression 

Notes

Acknowledgement

We are grateful to Donald L. Court (NCI-Frederick) for the generous gift of pSIM7 and to Dominick Matteau and Alain Lavigueur for critical reading of the manuscript. We thank the Centre de calcul scientifique of Université de Sherbrooke for computational resources and technical support. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). S.R. holds a Chercheur-boursier Junior 1 award from the Fonds de recherche Québec-Santé.

References

  1. 1.
    Esvelt KM, Wang HH (2013) Genome-scale engineering for systems and synthetic biology. Mol Syst Biol 9:641. doi: 10.1038/msb.2012.66 PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Marraffini L, Sontheimer EJ (2010) CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 11:181–190. doi: 10.1038/nrg2749 PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327:167–170. doi: 10.1126/science.1179555 CrossRefPubMedGoogle Scholar
  4. 4.
    Jiang W, Bikard D, Cox D et al (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239. doi: 10.1038/nbt.2508 PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Mali P, Esvelt KM, Church GM (2013) Cas9 as a versatile tool for engineering biology. Nat Methods 10:957–963. doi: 10.1038/nmeth.2649 PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32:347–355. doi: 10.1038/nbt.2842 PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278. doi: 10.1016/j.cell.2014.05.010 PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) PNAS Plus: Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci 109:E2579–E2586. doi: 10.1073/pnas.1208507109 PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–822CrossRefPubMedGoogle Scholar
  10. 10.
    Esvelt KM, Mali P, Braff JL et al (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods 10:1116–1121. doi: 10.1038/nmeth.2681 CrossRefPubMedGoogle Scholar
  11. 11.
    Anders C, Niewoehner O, Duerst A, Jinek M (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513:569–573. doi: 10.1038/nature13579 PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Nishimasu H, Ran FA, Hsu PD et al (2014) Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156:935–949. doi: 10.1016/j.cell.2014.02.001 PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Mali P, Aach J, Stranges PB et al (2013) Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31:833–838. doi: 10.1038/nbt.2675 CrossRefPubMedGoogle Scholar
  14. 14.
    Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. doi: 10.1126/science.1231143 PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Qi LS, Larson MH, Gilbert L et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183. doi: 10.1016/j.cell.2013.02.022 PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Bikard D, Jiang W, Samai P et al (2013) Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res 41:7429–7437. doi: 10.1093/nar/gkt520 PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Tanenbaum ME, Gilbert L, Qi LS et al (2014) A versatile protein tagging system for signal amplification in single molecule imaging and gene regulation. Cell 159:635–646. doi: 10.1016/j.cell.2014.09.039 CrossRefPubMedGoogle Scholar
  18. 18.
    Grenier F, Matteau D, Baby V, Rodrigue S (2014) Complete genome sequence of Escherichia coli BW25113. Genome Announc 2:e01038. doi: 10.1128/genomeA.01038-14 PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645. doi: 10.1073/pnas.120163297 PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Datta S, Costantino N, Court DL (2006) A set of recombineering plasmids for gram-negative bacteria. Gene 379:109–115. doi: 10.1016/j.gene.2006.04.018 CrossRefPubMedGoogle Scholar
  21. 21.
    Schleif R (2010) AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol Rev 34:779–796. doi: 10.1111/j.1574-6976.2010.00226.x CrossRefPubMedGoogle Scholar
  22. 22.
    Mathews DH, Sabina J, Zuker M et al (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288:911–940CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Frédéric Grenier
    • 1
  • Jean-François Lucier
    • 1
  • Sébastien Rodrigue
    • 1
    Email author
  1. 1.Département de Biologie, Faculté des SciencesUniversité de SherbrookeSherbrookeCanada

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