Genome editing using designer nucleases has revolutionized molecular biology by allowing DNA to be inserted, deleted or replaced in the genome of several model organisms. This protocol describes a marker-free co-selection strategy for high efficiency homology-driven and NHEJ-based gene editing in human cells by generating dominant cellular resistance to ouabain, a highly potent plant-derived inhibitor of the Na+,K+ ATPase (1, 2, 3). This scarless coconversion strategy is highly efficient and can yield a stable population of modified cells in 14 days. Techniques relative to sgRNA cloning, cell nucleofection, selection using ouabain and validation of gene disruption/insertion are described.