293T-EGFP cells were established by infecting 293T cells with lentivirus harboring an EGFP-expressing cassette. P4/RNP-mediated transfection was conducted by seeding into a glass-bottom cell well at a density of 104 cells per well the day before transfection. The doses of Cas9 protein, sgRNA, and P4 in each well were 2, 1, and 4 μg, respectively. After 4 hours of incubation, the Cas9-containing medium was replaced with fresh DMEM containing 10% FBS and incubated for 48 hours before analyzed by LSCM (ZEISS LSM 880, Germany). For flow cytometer analysis, 293T-EGFP cells were seeded into a six-well plate at a density of 2 × 105 cells per well. The doses of Cas9 protein, sgRNA, and P4 in each well were 4, 2, and 8 μg, respectively. After 48 hours of incubation, the cells were rinsed three times with 1 × PBS and trypsinized with 0.25% trypsin. Subsequently, the cells were resuspended in 200 μl of 1 × PBS and subjected to flow cytometry analysis (Beckman Coulter DxFLEX, USA).

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