Cell culture and transfection
The adherent CHO-K1 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) FBS (Hyclone). Cells were maintained as monolayer cultures in 25 cm2 T-flasks (Nunc) with a working volume of 5 ml. The serum free suspension adapted CHO-K1 cells were maintained in Power CHO medium (Lonza) supplemented with 8 mM L-Glutamine (Hyclone) and cultivated as suspension culture in 125 ml Erlenmeyer flasks (Corning) with a working volume of 20 ml. All cells were incubated in a humidified 5% CO2 at 37°C without shaking (adherent), or with 120 rpm shaking (suspension). Viable cell concentration was measured by a Countess II FL automated cell counter (Invitrogen) with the trypan blue dye exclusion method. Two transfection methods were used in this study; electroporation and lipofection. Electroporation was conducted using NEPA21 electroporator (Nepagene, Japan). According to the manufacturer’s instructions, adherent CHO-K1 cells were washed twice with Opti-MEM (Gibco) but not serum-free suspension CHO-K1. 1.0×106 cells were pelleted and re-suspended in 100 µl mixture of Opti-MEM (Gibco) and 10 µg DNA of Cas9/sgRNA expression vector and donor plasmid at a ratio 1:1 (w:w). Poring pulse and transfer pulse condition were as follows: 135 V, 7.5 miliseconds (ms) pulse length, two pulses, 50 ms pulse interval, 10% decay rate with + polarity and 20 V, 50 ms pulse length, five pulses, 50 ms pulse interval, 40% decay rate with +/- polarity respectively. After the electroporation, cells were immediately seeded in 6 well plates with pre-warmed culture media. Lipofection was conducted using Lipofectamine 3000 (Invitrogen) as per the manufacturer’s instructions.