Base pairing at the −2 and −3 positions does not interfere with CRISPR-immunity.

Abstract

<p><b>A</b>) Cells expressing g8<sup>C-2A</sup>-Cascade and Cas3 are partially resistant to M13 phage containing the CTT PAM adjacent to the g8 protospacer (white font/grey bar, e.o.p.∼10<sup>−2</sup>), but not when containing the CAT, CCT, CTC or CGT PAM (red font/red bars). Note that in the figure the PAMs are oriented in 3′ to 5′direction to display base pairing potential with the last three nucleotides of the crRNA repeat. The <i>in vitro</i> binding affinity of purified WT g8<sup>C-2A</sup>-Cascade for dsDNA containing the g8 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. <b>B</b>) Assays as in (A) using cells expressing the g8<sup>C-3G</sup> CRISPR, Cascade and Cas3, show that cells are not resistant to M13 phage containing the CAT, CTT, CCT, CTC or CGT PAMs adjacent to the g8 protospacer (red font/red bars). The <i>in vitro</i> binding affinity of purified WT g8<sup>C-3G</sup>-Cascade for dsDNA containing the M13 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. Asterisks indicate that the Kd value is >>1000 nM and the error bars represent the standard deviation of the mean.</p

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