Transfection of HEK293 FT Cells

HEK293 FT cells were transfected with CRISPR/Cas9 using Lipofectamine LTX Reagent with PLUS Reagent (15338100, Thermo Fisher Scientific, Grand Island, NY, USA). Briefly, HEK293 FT cells were plated into a 24-well plate. 750 ng plasmid was added to 50 μL Opti-MEM reduced serum medium (Thermo Fisher Scientific, Grand Island, NY, USA). Then 0.75 μL PLUS reagent was added, mixed, and incubated at room temperature for 5 min. 3 μL Lipofectamine in 50 μL Opti-MEM was then added and incubated at room temperature for 5 min. 100 μL DNA-lipid was added to one well. Transfection solution was distributed gently by moving the plate back and forth. Cells were harvested and sorted 48 hr later.