Figure 3.

The effect of CRISPR–Cas targeting on T7 infection. (A) The linear genome of bacteriophage T7 and positions of protospacers targeted by crRNA spacers of strains from our collection are schematically shown. The early genes region is colored black, the region containing middle genes is gray, late gene region is white. An area that contains both early and middle genes is striped. (B) EOP by T7 and EOT by cognate protospacer-containing plasmids into indicated strains. Mean values and standard deviations from three independent experiments with each strain are shown. EOP values for strains targeting additional protospacers can be found in Supplementary Table S1. (C) Growth curves of induced and uninduced cultures of T7-E-2-F infected with T7 at indicated MOIs. (D) Images of induced and uninduced T7-E2-F cultures infected with T7 at different MOIs. Images were taken at times indicated. Depolarized or lyzed cells appear red in the images. For each MOI, images in the top row show the progress of infection in the absence of cas gene expression; images at the lower row show infection of induced cells. (E) On the left, phage progeny production during the infection of induced and uninduced T7-L2-F cells. See Figure 1C legend for details. On the right, an agarose gel showing the products of SspI digestion of DNA prepared from uninfected, or infected (MOI = 2) induced and uninduced T7-L2-F cells collected 18 min post-infection. (F) Graphical representation of mapping of Illumina reads from total DNA prepared from T7-infected uninduced (top) or induced (bottom) T7-L2-F cells onto the T7 genome. Scale bars at the left show numbers of Illumina reads. The position of the protospacer targeted by T7-L2-F crRNA is shown at the bottom with a blue asterisk.