HEK293FT cells were transfected with LentiCRISPRv2Cre or pCREator EGFR WT, C1025A, L858R, and Δ8.2, and VSV-G plasmids in a 4:3:1 ratio using polyethylenimine. Twenty-four hours after transfection, the media were replaced with fresh DMEM supplemented with 25 mM Hepes (Gibco, 15630-080) and 3 mM caffeine (Sigma-Aldrich, C0750). Lentivirus-containing supernatant was collected from the cells at 48 and 72 hours following transfection, filtered through 0.45-μm filters (Thermo Scientific, 723-2545), and centrifuged at 107,000g. The viral pellet was soaked in 100 μl of phosphate-buffered saline (PBS) for 16 hours at 4°C, triturated, vortexed for 15 min at 4°C, and finally centrifuged at 16,000g for 30 s to remove insoluble debris. Lentivirus was then aliquoted and frozen at −80°C for later use. Lentivirus was titered on Green-Go cells, an NIH3T3 derivative harboring an integrated Cre-dependent GFP reporter. These cells were validated for reporter activity by flow cytometry during viral titering. A total of 2 × 105 cells were plated in six-well plates, and 24 hours later, lentivirus was added at 10, 1, and 0.1 μl per well. Cells were analyzed by flow cytometry for GFP expression 48 hours after infection, and viral titer was calculated accordingly.

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