The 293T cells, 293T-EGFP cells, HCT-116 cells, and HT-29 cells were cultured in DMEM (Gibco). The cell culture medium contains 10% FBS (Gemini), penicillin (100 μg/ml), and streptomycin (100 μg/ml) at 37°C under 5% CO2. P4/RNP-mediated transfection was conducted by seeding into a six-well plate at a density of 2 × 105 cells per well the day before transfection. The doses of Cas9 protein, sgRNA, and P4 in each well were 4, 2, and 8 μg, respectively. Six hours after transfection, transfection mixtures were replaced with DMEM containing 10% FBS and further incubated for 48 hours.

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