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CRISPR pp 349-362 | Cite as

Targeted Transcriptional Repression in Bacteria Using CRISPR Interference (CRISPRi)

  • John S. Hawkins
  • Spencer Wong
  • Jason M. Peters
  • Ricardo Almeida
  • Lei S. Qi
Part of the Methods in Molecular Biology book series (MIMB, volume 1311)

Abstract

Clustered regularly interspersed short palindromic repeats (CRISPR) interference (CRISPRi) is a powerful technology for sequence-specifically repressing gene expression in bacterial cells. CRISPRi requires only a single protein and a custom-designed guide RNA for specific gene targeting. In Escherichia coli, CRISPRi repression efficiency is high (~300-fold), and there are no observable off-target effects. The method can be scaled up as a general strategy for the repression of many genes simultaneously using multiple designed guide RNAs. Here we provide a protocol for efficient guide RNA design, cloning, and assay of the CRISPRi system in E. coli. In principle, this protocol can be used to construct CRISPRi systems for gene repression in other species of bacteria.

Key words

CRISPRi dCas9 sgRNA Escherichia coli 

Notes

Acknowledgements

We thank the Lei Qi lab, Carol Gross lab, and Wendell Lim lab for their support. J.S.H. acknowledges the support from Biophysics Graduate Program at UCSF. Spencer Wong acknowledges the support from Summer Research Training Program (SRTP) at UCSF. This work was supported by NIH P50 (grant GM081879, L.S.Q.), NIH Director’s Early Independence Award (grant OD017887, L.S.Q.), and a Ruth L. Kirschstein National Research Service Award (F32GM108222-01, J.M.P.).

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • John S. Hawkins
    • 1
    • 2
    • 3
  • Spencer Wong
    • 4
    • 8
  • Jason M. Peters
    • 5
  • Ricardo Almeida
    • 2
    • 3
    • 6
  • Lei S. Qi
    • 2
    • 3
    • 6
    • 7
    • 9
    • 10
    • 11
    • 12
  1. 1.Biophysics Graduate ProgramSan FranciscoUSA
  2. 2.Department of Cellular & Molecular PharmacologyUniversity of California, San FranciscoSan FranciscoUSA
  3. 3.UCSF Center for Systems and Synthetic BiologyUniversity of California, San FranciscoSan FranciscoUSA
  4. 4.Medical Microbiology and ImmunologyUniversity of California, DavisDavisUSA
  5. 5.Department of Microbiology and ImmunologyUniversity of California, San FranciscoSan FranciscoUSA
  6. 6.California Institute for Quantitative Biomedical ResearchSan FranciscoUSA
  7. 7.Center for Systems & Synthetic BiologyUniversity of California, San FranciscoSan FranciscoUSA
  8. 8.Department of BiologyMassachusetts Institute of TechnologyCambridgeUSA
  9. 9.Department of BioengineeringStanford UniversityStanfordUSA
  10. 10.Department of Chemical and Systems BiologyStanford UniversityStanfordUSA
  11. 11.Stanford ChEM-HStanfordUSA
  12. 12.Stanford UniversityStanfordUSA

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