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CRISPR pp 161-170 | Cite as

Exploring CRISPR Interference by Transformation with Plasmid Mixtures: Identification of Target Interference Motifs in Escherichia coli

  • Cristóbal Almendros
  • Francisco J. M. Mojica
Part of the Methods in Molecular Biology book series (MIMB, volume 1311)

Abstract

Plasmid transformation into a bacterial host harboring a functional CRISPR-Cas system targeting a sequence in the transforming molecule can be specifically hindered by CRISPR-mediated interference. In this case, measurements of transformation efficacy will provide an estimation of CRISPR activity. However, in order to standardize data of conventional assays (using a single plasmid in the input DNA sample), transformation efficiencies have to be compared to those obtained for a reference molecule in independent experiments. Here we describe the use of a transforming mixture of plasmids that includes the non-targeted vector as an internal reference to obtain normalized data which are unbiased by empirical variations.

Key words

CRISPR-Cas systems Electroporation Plasmid transformation Interference efficiency Escherichia coli Target interference motif Protospacer adjacent motif 

Notes

Acknowledgements

This work was supported by a grant from the Spanish Ministerio de Economıía y Competitividad (BIO2011-24417). We thank Arlette Darfeuille-Michaud (Clermont Université, Université d’Auvergne, France) for strain LF82.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Cristóbal Almendros
    • 1
  • Francisco J. M. Mojica
    • 1
  1. 1.Departamento de Fisiología, Genética y MicrobiologíaUniversidad de AlicanteAlicanteSpain

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