Human pluripotent stem cells (hPSCs) represent a formidable tool for disease modeling, drug discovery, and regenerative medicine using human cells and tissues in vitro. Evolving techniques of targeted genome editing, specifically the CRISPR/Cas9 system, allow for the generation of cell lines bearing gene-specific knock-outs, knock-in reporters, and precise mutations. However, there are increasing concerns related to the transfection efficiency, cell viability, and maintenance of pluripotency provided by genome-editing techniques. The procedure presented here employs transient antibiotic selection that overcomes reduced transfection efficiency, avoids cytotoxic flow sorting for increased viability, and generates multiple genome-edited pluripotent hPSC lines expanded from a single parent cell. Avoidance of xenogeneic contamination from feeder cells and reduced operator workload, owing to single-cell passaging rather than clump passaging, are additional benefits. The outlined methods may enable researchers with limited means and technical experience to create human stem cell lines containing desired gene-specific mutations.