"Functionality of C. difficile cas genes for plasmid interference in E. coli. The transformation efficiency was estimated with pT7Blue derivative plasmids carrying the wild-type (wt) protospacer corresponding to the first spacer of the CRISPR 16 array (CR16) (rows 2 and 5) or a mutated protospacer CR16 (rows 3 and 6) compared to the pT7Blue empty vector used as a negative control (rows 1 and 4). The protospacer plasmid used is indicated to the left of the photographs together with schematic representation of E. coli strains carrying engineered CRISPR arrays with the corresponding spacer under the control of T7 RNAP promoter (T7). E. coli KD623 strain (rows 1 to 3) carries C. difficile CRISPR ""miniarray"" with the first spacer of CRISPR 16 array flanked by repeats, and E. coli KD626 strain (rows 4 to 6) carries reduced ""miniarray"" with one repeat lacking spacer sequence. The CRISPR ""leader"" region (LDR) is indicated. Both strains were transformed with pCDF1-b vector derivative, allowing the expression of C. difficile cas gene set lacking cas1 and cas2 (from CD2982 to CD2977). The Cas protein production and crRNA expression were induced by the addition of 1 mM l-arabinose and 1 mM IPTG. The serial dilutions of transformation mixtures deposited on LB plates with ampicillin are indicated (ND, not diluted)."