Co-transfection

Three plasmids (an FP donor vector, a frame selector Cas9/sgRNA vector and a target Cas9/sgRNA vector) were transfected into mESCs using Lipofectamine 2000 (Thermo Fisher Scientific, 11668027). The molar ratio of [donor vector]:[frame selector]:[target sgRNA] plasmid was typically 20:1:1, and was achieved using 364 ng minicircle donor, 130 ng target Cas9/sgRNA vector and 130 ng frame selector. The three plasmids were mixed and diluted to 50 µl in Opti-MEM Medium (Gibco, 31985062). 1.25 µl Lipofectamine 2000 was diluted to 50 µl in Opti-MEM Medium, then added to the 50 μl of DNA solution and incubated at room temperature for 5 min. After incubation, the 100 μl DNA/Lipofectamine 2000 mixture was transferred to a gelatin-coated 12-well plate. 5×10⁵ single cells in suspension per well were added to DNA/Lipofectamine 2000 mixture. 2i/LIF was added up to 1 ml per well. The same procedures were performed with 50 μl of pure Opti-MEM Medium and 50 μl of diluted Lipofectamine 2000 as a negative control. The transfection mixture was replaced with 2-3 ml of fresh 2i/LIF and mCherry-Cas9 signal observed in the transfected cells the following day. Triplicates of co-transfection were used for each gene tagging. Table S4 lists plasmids that were co-transfected in each experiment.