Assessing the sensitivity of SENECA to detect primed adaptation in the type I-E CRISPR-Cas system of E. coli.
(A) To create an E. coli strain suitable for detection of primed adaptation by SENECA, parental E. coli KD263 cells containing inducible cas genes and a CRISPR array with a single spacer Sg8 are transformed with a plasmid carrying a priming protospacer PPSg8 matching Sg8 and an FaqI site introduced in a previously characterized highly used ‘hot’ protospacer (PSFaqI) shown as a turquoise rectangle. Upon the recognition of PPSg8 by the Cascade-crRNA effector complex, primed adaptation occurs and new spacers originating from the plasmid are integrated into the CRISPR array [31,41,65]. Among colonies that lost the plasmid and expanded their CRISPR array (see Figure 1B), a clone that acquired the SFaqI spacer is selected. (B) Genomic DNA purified from parental cells carrying the SFaqI spacer or a derivative with an expanded array (+1) was mixed at ratios indicated and the state of CRISPR arrays was assessed by standard PCR (see Figure 2A) or SENECA (see Figure 2D). The percentage of the +1 genomic DNA in the sample is indicated above the gel. At the left-hand side, amplicons corresponding to unexpanded and expanded arrays revealed by standard PCR are labeled +0 and +1, correspondingly. The expanded array amplicon generated by SENECA is also labeled +1. The lower minor band (gray triangle) corresponds to the results of linear amplification of the parental array [59].