Protospacer, PAM, and repeat mutants can escape toxicity.

<p>(A) Predicted pairing between an <i>expI</i> crRNA and the <i>expI</i> protospacer (PAM is green, spacer is blue, protospacer is red and seed sequence is bold and underlined). (B) Left; protospacer sequences in WT, Δ<i>expI</i> (PCF81), Δ<i>expI</i> with a single WT (RBV01), C3T (RBV04), C6T (RBV03) or G-1T (PAM) (RBV02) <i>expI</i> protospacer. Right; toxicity assays of a single anti-<i>expI</i> spacer (<i>expI</i>; pE1-16; white bars) or a plasmid with no spacers (none; pC1-16; black bar) when expressed in the backgrounds shown on the left. (C) Predicted folds and position of mutations for WT, G20A, C18A and C18A/G8U single CRISPR repeats. The black triangle represents the site of cleavage by Cas6f. (D) Toxicity assays of plasmids expressing a single anti-<i>expI</i> spacer flanked by either WT (pE1-16), G20A (pE1-16 G20A), C18A (pE1-16 C18A) and C18A/G8U repeats (pE1-16 C18A/G8U) in WT <i>P. atrosepticum</i>.