a, Cloning strategy for spacer insertion into the selection plasmid (plasmid no. 1, Fig. 1b). Spacer inserts are generated via annealing of single strand oligos with overhangs matching the BsmBI restriction digest site followed by Golden gate assembly into the empty selection plasmid. b, Test of spacers targeting JS26. Efficiency of plating (EOP) of JS26 lysate on P. confusarubida following a 5 h infection of a non-targeting control (eV ctl) at multiplicity of infection (MOI) = 1, compared with infection of targeting strains expressing either a single spacer targeting JS26 (spacer A & B) a two-spacer array comprising spacers in either orientation (double spacer A-B & B-A), or a mixed culture (1:1) consisting of a strain expressing spacer A and a strain expressing spacer B (mixed culture A + B). When no single plaques were visible, EOP was calculated as one plaque in the first dilution that did not show any clearing of the bacterial lawn. Data are the geometric mean of n = 3 independent biological replicates ± geometric s.d., and individual data points are shown. c, Plaque PCR testing for successful transposon insertion. Following transposon mutagenesis of JS26 (n = 3 independent biological replicates), phage lysates were plated...

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