Our workflow for knocking in AcGFP1 under the control of the EF1α promoter into the AAVS1 locus began with hiPS cells cultured in our Cellartis DEF-CS 500 Culture System, which provided a homogeneous, undifferentiated starting population. We used electroporation to deliver Cas9-sgRNA together with the HDR template. We delivered the Cas9-sgRNA complex in the form of ribonucleoprotein (RNP) in order to decrease off-target effects and for footprint-free genome editing. We used our own system to synthesize a long ssDNA donor template that has a reduced tendency to randomly integrate and a low cytotoxic response, as compared to dsDNA. From the overall edited population, AcGFP1+ cells were single-cell isolated by limiting dilution and expanded to generate clonal cell lines.