Library preparation and CRISPR screen

IMCD-3 cells were transduced with lentivirus containing Cas9 (pXPR_101, Addgene #52962) by spinoculation (1 mL of Cas9 lentivirus onto 3 × 10⁶ cells), then selected with 5 μg/mL blasticidin (ThermoFisher A1113903). Cas9 activity was assayed by transducing cells with lentivirus containing pXPR_011 (Addgene #59702), which expresses eGFP and a sgRNA targeting eGFP, and selecting with 5 μg/mL puromycin (ThermoFisher A1113803). Loss of GFP expression in cells expressing the eGFP guide was assessed by flow cytometry as described above. After Cas9 activity was confirmed, a Brie mouse sgRNA library (Brie pXPR_003, Batch 3, Lot #m-AA89-20171107; generated by the Broad Institute) was transduced at MOI 0.5 into IMCD-3 Cas9 cells by spinoculation and selected with 5 μg/mL puromycin for 10 d until screening.

For screening, 5 × 10⁶ IMCD-3 cells were seeded into eighty 15-cm dishes the day before infection. Cells were infected with UTI89 at MOI 150 and incubated at 37°C for 45–75 min, then fixed and stained for flow cytometry as described above. Samples were placed at 4°C overnight on a tube roller; the following day, the 5% least FITC-positive cells were collected on a Sony iCyt Synergy BSC sorter. Tubes containing these low-FITC sorted cells were centrifuged, resuspended in 250 μl PBS, and stored at -20°C. A total of 6 × 10⁸ mock-treated cells were harvested, separated into aliquots of 8 × 10⁷ cells, resuspended in 2 mL PBS, and stored at -20°C until genomic DNA preparation. DNA was extracted using QIAamp DNA Blood Maxi kit (Qiagen 51192) for mock samples and QIAamp DNA FFPE Tissue kit (Qiagen 56404) for experimental samples.