Optimization of crRNA concentration and peroxisome relocalization a ssay. (A) Quantification of CRISPR/Cas9 editing frequency in ARPE-19 and U-2 OS cells with varying crRNA concentration. Cells were transfected in a 384-well format with mRNA-Cas9 (40 ng/well) coupled with MessengerMAX (1%; vol/vol) for 6 h before transfecting with a panel of six gene-targeting crRNA pools (crLIS1, crDYNC1H1, crPPIB, crDCTN1, crGOLGA3, and crTARDBP) or the NTC pool at the indicated concentrations. The Tris buffer used for RNA resuspension was used as an additional control. Cells were fixed 72 h later and stained with antibodies to the corresponding protein products to evaluate the frequency of target protein depletion (gating based on the range of fluorescence signal of NTC cells). A final concentration of 50 nM crRNA per well was selected for the genome-wide screen. (B) Representative results of applying a spot detection mask on raw images of rapamycin-treated U-2 OS PEX cells. Scale bar, 50 µm. (C) Optimization of rapamycin concentration and treatment duration in the peroxisome relocalization assay. Note that, with these raw values, the number of GFP spots increases with perinuclear clustering because discrete puncta are otherwise relatively uncommon due to the dim signal, whereas RFP spot number decreases with perinuclear clustering as signals from multiple dispersed puncta coalesce at the MTOC. A 2.5-h treatment with 2 nM rapamycin was selected for the genome-wide screen. (D) Impact of the number of seeded cells per well (384-well plate format) on the number of GFP-BICD2N-FRB and PTS-RFP-FKBP spots. Cells were seeded for 72 h prior to treatment with rapamycin (2 nM). 1,500 cells per well were seeded for the genome-wide screen. In A, C, and D, data points represent mean aggregation at well level (minimum of 100 cells from at least two wells analyzed per condition). Error bars, SD.