The Southern blots obtained with NsiI and KpnI-digested genomic DNA from four out of the five above-mentioned clones, and untargeted hESCs DNA as control, clearly revealed single-copy integration of the seamless cassette for each clone/locus (Fig. 3a) and confirmed the stable integration of the seamless vector in intron 2 of CDCA7L (Cell Division Cycle Associated b 7 Like; Chr7) in clones F1 and F9, intron 4 of CCDC141 (Coiled-Coil Domain Containing 141, Chr2) in clone B6 and intron 7 of DMD (Duchenne Muscular Dystrophy, ChrX) in clone B8 (Fig. 3a, b, Supplementary Table 2). This evidence concerns the gene DMD and Duchenne muscular dystrophy.