Figure 1.

The application of Cas9^FCPF for live cell imaging. (A) Overview of applications of Cas9^FCPF for labeling (FITC-FCPF, green) and degradation (PROTAC-FCPF, orange). (B) Green fluorescence signal is detected in FCPF-FITC-treated HeLa cells expressing Cas9^FCPF. HeLa cells expressing Cas9^FCPF were treated with FITC-FCPF (10 μM) for 2 h. Cas9^GFP was used as a positive control. The fluorescence images (10× and 63× enlarged) were taken from nontransfected cells, FITC-FCPF-treated cells expressing Cas9^WT, and nontreated cells expressing Cas9^FCPF. Images from Cas9^GFP cells and Cas9^FCPF cells treated with FITC-FCPF (10 μM, 2 h) were shown. Remaining images can be found in the Supporting Information (Figure S1A). Scale bar: 40 μm. (C) Comparison of fluorescence intensity of FITC-FCPF-treated cells expressing Cas9^WT and Cas9^FCPF HeLa cells expressing Cas9^WT or Cas9^FCPF were treated with FCPF-FITC (10 μM) for 2 h, and the fluorescence intensity was analyzed by FACS. Nontransfected cells and cells expressing Cas9^GFP were used as negative and positive controls. The results were obtained from four independent biological replications. The center represents the median. Two-way ANOVA was performed. *** p < 0.001. (D) Electrophoresis analysis of FITC-FCPF-treated samples obtained from cells expressing either Cas9^FCPF or Cas9^WT. HeLa cells expressing Cas9^WT or Cas9^FCPF were treated with increasing concentrations of FITC-FCPF for 2 h. Proteins collected from a whole cell lysis were dissolved in 8% SDS PAGE. The original pictures with a protein loading marker can be found in the Supporting Information (Figure S1C).