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)


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 



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.


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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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