Figure 2.

The effect of CRISPR–Cas targeting on T5 infection. (A) The linear genome of bacteriophage T5 and positions of protospacers targeted by crRNA spacers of strains from our collection are schematically shown. Pre-early genes are colored black, the region containing early genes is gray, late gene region is white. The T5 genome is terminally redundant with the entire pre-early area present at either end of the genome. Spacers targeting pre-early region thus have two matching sites. Protospacers at the right end are shown as grey arrows. Protospacers originating from different strands of the genome are shown above or below the genome scheme. (B) EOP by T5 and EOT by cognate protospacer-containing plasmids into indicated strains. Results obtained with pre-early targeting strains are shown on the left. Results obtained with early and late targeting strains are on the right. Mean values and standard deviations from three independent experiments with each strain are presented. EOP values for strains targeting additional protospacers can be found in Supplementary Table S1. (C) Growth curves of induced (‘+Ind’) and uninduced (‘–Ind’) cultures of T5-PE-6-R (left) and T5-E4-F (right) strains infected with T5 at indicated MOIs. (D) Phage progeny production during the infection of T5-PE-6-R (left) or T5-E4-F (right) cultures. See Figure 1C legend for details. At the right of each diagram with growth curves, agarose gels showing the products of SalI and MluI digestion of DNA prepared from cells from an uninfected culture, or induced and uninduced infected (MOI = 2) cultures collected 45 min post-infection are presented. Phage DNA restriction fragments are marked by asterisks. (E) Graphical representation of HTS results of spacers acquired by the T5-PE-6-R cultures infected with the A2G escape T5 phage. Red vertical lines indicate spacers matching the non-target strand, blue lines—spacers matching the target strand. Line heights indicate relative frequency of reads corresponding to each acquired spacer. Scale bar at the left shows numbers of Illumina reads. The pre-early region at the left end of the genome is expanded below to show mapping results at a larger scale. The position of the priming protospacer is indicated. A vertical black line shows the position until which phage DNA is initially inserted into the infected cell (21,22). Rectangular bars show the proportion of spacers matching non-target (red) and target (blue) strands upstream (‘U’) and downstream (‘D’) of the priming protospacer and the total strand bias of acquired spacers (‘U+D’).