Fig 5.

Upstream genomic regions are important for BFL-1 transcription, and BFL-1 protects against extrinsic apoptosis. (A) Schematic depicting TRE-dCas9-KRAB experimental system. Doxycycline (Dox) addition induces the expression of dCas9-KRAB but requires sgRNA expression from the lentiguide-puroR system to target the genomic DNA. (B) qPCR performed on LCLs transduced with TRE-dCas9-KRAB with or without sgRNAs targeting the BFL-1 TSS and upstream regions. Cells were treated with 3 mg/mL Dox for 48 hours. (C) Dox-treated cells are washed and then treated for 48 hours. Mean and SEM are plotted and normalized to untreated samples, with significance determined through one-way ANOVA with multiple comparisons to “No sgRNA” control. (D) DNA gel showing representative PCR confirming Cas9-mediated editing of the RBS region. PCR was performed on genomic DNA isolated from donor-matched WT and Δ3A LCLs and wild-type LCLs 5 days post-transfection with Cas9 coupled with sgRNAs targeting the AAVS1 site, sg1 and sg2 of the RBS, and combined sg1 and sg2. (E) qPCR performed on WT and Δ3A LCLs and WT LCLs transfected with Cas9 RNPs targeting the RBS. Results are from four separate transfections on two separate days, day 5 post-transfection. Significance by one-way ANOVA, multiple comparisons, and mean and SEM reported. *P < 0.05, ***P < 0.005.