Potential base pairing between the crRNA repeat regions and protospacer flanking regions does not affect CRISPR-interference.

Abstract

<p><b>A</b>) Model of the R-loop formed by Cascade during dsDNA binding. <b>B</b>) Cells expressing WT g8-Cascade and Cas3 are resistant to plasmids containing the CAT PAM adjacent to the g8 protospacer (black bars, transformation efficiency 6.7±1.5×10<sup>5</sup> cfu/µg DNA for plasmid pWUR690 and 6.8±0.9×10<sup>5</sup> cfu/µg DNA for plasmid pWUR688), but are susceptible to plasmid transformation when the g8 protospacer is flanked by a CGG PAM, which is fully complementary to the 5′-handle (red bars, transformation efficiency 4.2±0.9×10<sup>8</sup> cfu/µg DNA for plasmid pWUR687 and 4.5±0.8×10<sup>8</sup> cfu/µg DNA for plasmid pWUR689). Transformation efficiency for a control pUC19 plasmid is 6.2±1.1×10<sup>8</sup> cfu/µg DNA. The histogram shows the <i>in vitro</i> binding affinity of purified WT g8-Cascade for dsDNA containing the g8 protospacer flanked by sequences with a varying base pairing potential, as shown on the right. 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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