Southern blot analysis of ΔpksP mutant generated in the Af293 background. Southern blot analysis of ΔpksP mutant generated in the Af293 background. (A)

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Southern blot analysis of ΔpksP mutant generated in the Af293 background. Southern blot analysis of ΔpksP mutant generated in the Af293 background. (A) Schematic representation of the genomic locus of the Af293 and ΔpksP strains. Deletion of the pksP gene was carried out using the HygR cassette. The cleavage sites of the dual in vitro-assembled Cas9 RNPs are marked by thick vertical lines. XhoI cutting sites are indicated in the pksP locus of the wild-type and ΔpksP strains. (B) Southern blot analysis of 6 arbitrarily selected colonies after digesting genomic DNA with the XhoI restriction enzyme. The wild type (WT) produced a 1.8-kb band that matches the expected wild-type banding pattern. Lanes 1, 2, 4, 5, and 6 displayed a 3.8-kb band which matches the expected pksP deletion banding pattern. The colony in lane 3 displayed a 7.6-kb band, likely containing a tandem integration of the HygR repair template at the pksP locus. Qusai Al Abdallah et al. mSphere 2017; doi:10.1128/mSphere.00446-17