title: Exploring CRISPR Interference by Transformation with Plasmid Mixtures: Identification of Target Interference Motifs in Escherichia coli.
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.
authors.affiliation: Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Pavilion 12, 03080, Alicante, Spain.
authors.forename: Cristóbal
authors.initials: C
authors.lastname: Almendros
authors.forename: Francisco J M
authors.initials: FJ
authors.lastname: Mojica
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics*; D059372:Nucleotide Motifs / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D014169:Transformation, Bacterial