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. 2022 Jun 7;50(11):6414–6422. doi: 10.1093/nar/gkac462

Figure 1.

Figure 1.

Cas12a targeting does not elicit abortive infection or prevent the rise of phage escapers. (A) Growth curves of P. aeruginosa PAO1 tn7::LbCas12a strain expressing targeting or non-targeting crRNAs infected with ϕDMS3 at multiplicities of infection (MOI) indicated in legend. (B) In vitro LbCas12a cis- and trans-cleavage assay with dsDNA template (plus or minus PAM) and ssDNA. dsDNA template encodes same protospacer encoded in ϕDMS3 for experiments in (A). (C) Phage plaque assays with ten-fold serial dilutions of wildtype or mutated ϕ JBD30 phage. Bacterial clearance (black) indicates phage replication. Sequences of the targeted phage gene show mutated nucleotides (magenta) in phages able to escape Cas12a targeting. (D) Phage plaque assays in which targeting and non-targeting strains were infected with ten-fold serial dilutions of escaper phage alone or with escaper phage and a high titer of targeted wildtype phage. PAM, protospacer adjacent motif (teal); WT, wildtype phage; ESC, escaper phage. NT, non-target due to lack of PAM; T, target with PAM.