Regulation of Cas9 activity by BOC in mammalian in vitro cell culture. (A) Immunoblotting (IB) of HEK293 cells co-transfected with a construct expressing the HA-tagged Cas9 mutant indicated plus another construct encoding eGFP-DD, PylRS, gRNA and Pyl tRNA. Transfectants were incubated in the presence (+) or absence (−) of 1 mM BOC for 24 h before protein detection with antibodies against HA (Cas9) or GFP. Upper and lower panels represent cropped regions of the same representative (n = 3) immunoblot, whose corresponding uncropped, full-length versions are presented in Supplementary Figure S3B. (B) Induction of Cas9K510B following transient transfection of HEK293 cells and incubation for 24 h in the presence of the indicated concentrations of BOC. Induction was inferred by determining protein levels by immunoblotting with antibodies against HA (Cas9K510B) or GAPDH (GAP) and HA/GAP ratios normalized (value = 1.0) against the ratio at 1,000 μM BOC. nd, not detected. (C) Stability of Cas9 variants in HEK293 cells. Cells were transfected with constructs for PylRS, PylT, Cas9K510B or Cas9K510B-DD and cultured in the presence of 1 mM BOC for 24 h. Transfectants were then washed with DMEM three times and analyzed after a further 0, 24 or 48 h by immunoblotting (IB) with antibodies against HA (for Cas9, which has an HA-tag) or a GADPH (GAP) loading/transfer control. Upper (full length) and lower (cropped) panels correspond to the same representative (n = 3) immunoblot. (D) Plots of data from (C), in which the ratio of HA/GADPH signals at 0 h was set to 1.0 for each series. Error bars (s.d.) were from experimental triplicates. For the difference between Cas9K510B or Cas9K510B-DD after 48 h, p = 0.002 (n = 3).