Reverse Transfection of Synthetic gRNA

NCI-H358-Cas9 cells were seeded at 2.75 × 10⁵ cells/cm² and grown for 3 d prior to induction of Cas9 with doxycycline (100 ng/mL) addition 24 h before reverse transfection. Duplexed cr:tracRNA was acoustically dispensed with an Echo 555 (Labcyte) into 384-well assay plates (Cell Carrier Ultra, PerkinElmer, Waltham, MA) followed by the addition of transfection solution (10 µL serum-free RPMI-1640, 0.6 % [v/v] RNAiMAX, Thermo Fisher Scientific). After a 20-min incubation at RT, 40 µL cell suspension was dispensed into assay plates (4000 cells/µL) and incubated at 37 °C, 5% CO₂.