Figure 6. Csm6 is required to provide immunity against viruses with target mutations.

(A) Introduction of mutations (in red) in the spacer targeting the gp43 phage gene that generate 3, 4 or 5 mismatches in the crRNA:target region. (B) Staphylococci harboring a wild-type III-A CRISPR-Cas system targeting the gp43 gene in the presence of different crRNA:target mismatches were grown in liquid media and infected with ϕNM1γ6 phage (at 0 hours) with a multiplicity of infection of 5 viruses per bacteria. Optical density at 600 nm (OD₆₀₀) was measured for the following 12 hours to monitor cell survival. Representative growth curves of at least three independent assays are shown. (C) Same as panel (B) but with cells harboring a CRISPR-Cas locus without csm6. (D) qPCR performed on the ΦNM1γ6 gp43 gene using total DNA collected at different times post-infection from cells carrying CRISPR-Cas systems targeting in the presence (4 mm) or absence (0 mm) of crRNA:target mismatches. Values for the rho gene were used for normalization to obtain the relative abundance of the gp43 gene for each data point (mean ± S.D. of four replicas). (E) pTarget plasmid DNA, harboring the gp43 target under the control of a tetracycline-inducible promoter, was extracted from cells harboring a type III-A CRISPR-Cas system without a spacer (Δspc) or with a gp43-targeting spacer with or without mismatches (4 mm or 0 mm, respectively), at different times after treatment with aTc. Plasmid DNA was visualized by agarose gel electrophoresis followed by ethidium bromide staining. (F) The gp32 spacer has a complete match in the ϕNM1γ6 genome but presents four mismatches in the ϕNM4γ4 phage. (G) Staphylococci harboring a wild-type, Δcsm6 or Δspc type III-A CRISPR-Cas system with the gp32 spacer were grown in liquid media and infected with ϕNM1γ6 phage (at 0 hours) with a multiplicity of infection of 5 viruses per bacteria. Optical density at 600 nm (OD₆₀₀) was measured for the following 12 hours to monitor cell survival due to CRISPR immunity against the phage. Representative growth curves of at least three independent assays are shown. (H) Same as (G) but following infection with phage ϕNM4γ4. See also Fig. S4.