Figure 2. pRAM18-Tet-On can be used to express dCas9 in R. parkeri.
(A) Schematic of pRAM18-based CRISPRi system. Expression of dCas9 is driven by the Tet-On promoter and the sgRNA is driven by the constitutive promoter PrpsL.
(B) Four dCas9 variants were cloned into pRAM18dSGA. Each dCas9 variant recognizes a distinct PAM, with each PAM found in varying instances in the R. parkeri genome.
(C) Expression of dCas9 in R. parkeri. Each dCas9 variant was tagged with a C-terminal HA epitope and expression −/+ aTc was visualized by Western blot, as well as OmpA as a loading control.