Figure 3.

The PROTAC-FCPF-induced Cas9^FCPFprotein degradation is ubiquitin-dependent. (A) MG132 inhibits PROTAC-FCPF-mediated Cas9^FCPF protein degradation. HeLa cells expressing Cas9^WT or Cas9^FCPF were treated with PROTAC-FCPF (10 μM) in the absence or presence of MG132 (5 μM) for 8 h. DMSO was used as a control. Cas9 protein was immunoprecipitated, and immunoblotting was performed for detecting the ubiquitination. (B) Comparison of PROTAC-FCPF-induced Cas9^FCPF in CRBN-WT or CRBN-KD HeLa cells. HeLa cells were cotransfected with Cas9^FCPF and control siRNA or Cas9^FCPF and CRBN siRNA for 48 h and treated with increasing concentrations of PROTAC-FCPF for 8 h. The control experiment in cells expressing Cas9^WT can be found in Figure S3B. (C) PROTAC-FCPF-mediated Cas9^FCPF protein degradation requires functional CRBN. (upper) Illustration of in vitro ubiquitination assay. In vitro degradation was performed using the whole CRBN-KO cell lysate, in which either FLAG-Cas9^WT or FLAG-Cas9^FCPF was expressed. FLAG-CRBN^WT and FLAG-CRBN^m were purified separately. The in vitro degradation was initiated by adding FLAG-CRBN into the cell lysate and incubated for 90 min at 37 °C in the presence or absence of PROTAC-FCPF (100 μM). The residual Cas9 protein was compared using immunoblotting, or (D) the ATP consumption was measured after an incubation with PROTAC-FCPF (100 μM) or the combination of PROTAC-FCPF (100 μM) and lenalidomide (100 μM) at 37 °C for 90 min. The result was obtained from at least three independent biological replications with at least two technical replications of each. Two-way ANOVA was performed. (E) PROTAC-FCPF degrades Cas9^FCPF in cells lacking VHL but requires a DDB1 expression. HeLa cells were cotransfected with Cas9^FCPF and DDB1 siRNA or VHL siRNA for 48 h and treated with PROTAC-FCPF (10 μM) for 8 h. (F) PROTAC-FCPF-mediated Cas9^FCPF degradation was rescued in the presence of PYR41, an E1 inhibitor (50 μM), or MLN4924, an NEDD8-activating E1 enzyme (10 μM). HeLa cells expression of Cas9^FCPF were treated with PROTAC-FCPF (10 μM) or in the combination of PYR41 or MLN4924 for 8 h. (G) Evaluation of specificity of PROTAC-FCPF-mediated Cas9^FCPF degradation by a proteome-wide analysis. HeLa cells expressing either Cas9^WT or Cas9^FCPF were treated with PROTAC-FCPF (10 μM) for 6 h. DMSO was used as a mock. High specificity of PROTAC-FCPF-mediated Cas9^FCPF degradation was reproducible from three independent experiments. Eight of a total of 4436 protein are highlighted due to their significant alternations in PROTAC-FCPF-treated cells expressing Cas9^FCPF as compared with those in Cas9^WT cells. Cas9 was highlighted in red. Detailed information regarding the experiment protocol and an analysis of proteomics can be found in Materials and Methods.