A single nucleotide PAM mutation enables escape from native CRISPR/Cas targeting.

<p>(A) <i>P. atrosepticum</i> CRISPR/Cas genomic organisation. The <i>cas1</i> and the <i>cas2</i>-<i>cas3</i> hybrid genes are shown in blue, <i>csy1</i>-<i>3</i> in pale blue, <i>cas6f</i> in orange and the CRISPRs as grey arrows in the direction of transcription. CRISPR2 and 3 are separated by a toxin/antitoxin system (white arrows). (B) Spacer 6 of CRISPR2 (from leader) contains deviations from the repeat consensus (shown in blue) and (C) has a 100% match to a protospacer (red) within <i>eca0560</i> in HAI2 in the <i>P. atrosepticum</i> genome. The protospacer matching spacer 6 contains a non-consensus 5′-protospacer-TG-3′ PAM (green). (D) Toxicity assays in the WT with plasmids containing consensus repeats and either no spacer (pC1-16), a single native spacer 6 (5′-protospacer-TG-3′ PAM; pTraGS6-16) or an engineered spacer (5′-protospacer-GG-3′ PAM; pTraG1-16) that targets <i>eca0560</i>. The protospacer locations in <i>eca0560</i> of the native (pTraGS6-16) and engineered (pTraG1-16) spacers are shown below in red. (E) Sequence and pairing of the engineered anti-<i>eca0560</i> spacer (in pTraG1-16) with the consensus 5′-protospacer-GG-3′ PAM (green).