Fig. 3

Influence of spacer length on DNA interference. a Constructs of altered spacer length. WT Cascade contains spacer of 32 nt length, which was truncated or extended by increments of 6 nt at the spacer’s 3′-end. Cascade complexes are named according to the alteration of the WT spacer length. Target DNA bearing CC PAM and protospacer, matching to all spacers, is depicted below. Schematic representation of WT, −12 and +12 Cascade complexes is shown on the right. b Phage and c plasmid interference mediated by altered-length Cascade complexes. Phage interference is represented as ratios of PFU of M13-NT (non-target) and M13-SP-CC (target) phages in the context of Cascade complexes only. Plasmid interference is depicted as transformation efficiencies (CFU/μg of DNA) of pNT (non-target) or pSP-CC (target) plasmid DNA in the background of the complete Aa-CRISPR-Cas system. d Composition of purified Cascade complexes. SDS-PAGE of purified protein preparations is presented on the top. Denaturing polyacrylamide gel (on the bottom) shows extracted crRNA from the altered Cascade complexes. e Binding of altered-length Cascade complexes to DNA. Binding of Cascade variant to non-target (NT; on the top) or target (SP-CC; on the bottom) DNA was monitored by EMSA. f Binding affinities of altered-length Cascade complexes to target and non-target DNA. Dissociation constant K_d values were obtained by EMSA. g Triggering of target DNA degradation by altered-length Cascade complexes. Cleavage of the linearized plasmid (pSP-CC) was monitored in the presence of Cas2/3 and Cascade variant. Error bars in the b, c, and f represent standard deviations in at least three separate experiments (individual data values are provided in Additional file 14)