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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mojica FJM, Garrett RA (2013) Discovery and seminal developments in the CRISPR field. In: Barrangou R, van der Oost J (eds) CRISPR-Cas systems: RNA-mediated adaptive immunity in bacteria and archaea. Springer, Berlin
Shah SA, Erdmann S, Mojica FJM, Garrett RA (2013) Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol 10:891–899
Brouns SJ, Jore MM, Lundgren M et al (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964
Zabarovsky ER, Winberg G (1990) High efficiency electroporation of ligated DNA into bacteria. Nucleic Acids Res 18:5912
Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145
Shi X, Karkut T, Alting-Mees M et al (2003) Enhancing Escherichia coli electrotransformation competency by invoking physiological adaptations to stress and modifying membrane integrity. Anal Biochem 320:52–155
Almendros C, Guzmán NM, Díez-Villaseñor C et al (2012) Target motifs affecting natural immunity by a constitutive CRISPR-Cas system in Escherichia coli. PLoS One 7:e50797
Miquel S, Peyretaillade E, Claret L et al (2010) Complete genome sequence of Crohn’s disease-associated adherent-invasive E. coli strain LF82. PLoS One 5:e12714
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Westra ER, van Erp PBG, Kunne T et al (2012) CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol Cell 46:595–605
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Almendros, C., Mojica, F.J.M. (2015). Exploring CRISPR Interference by Transformation with Plasmid Mixtures: Identification of Target Interference Motifs in Escherichia coli . In: Lundgren, M., Charpentier, E., Fineran, P. (eds) CRISPR. Methods in Molecular Biology, vol 1311. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2687-9_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2687-9_10
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2686-2
Online ISBN: 978-1-4939-2687-9
eBook Packages: Springer Protocols