Strategy for seamless correction of the β-thalassemia mutations using piggyBac and Cas9. (A) Locations of the two mutations at −28 with A/G substitution and the codon 41/42 with 4-bp deletion. (B) The DSB at intron 1 following Cas9 cleavage. (C) The targeting construct of the piggyBac transposon carrying the selectable markers, puro∆tk and Neo, flanked by 500 bp of wild-type genomic sequences. (D) Insertion of the piggyBac following homologous recombination. (E) After selection with puromycin, clones with mutation-corrected lines were identified and transiently transfected with transposase expression plasmids, followed by treatment with FIAU to eliminate piggyBac-containing clones, and the seamless mutation-corrected clones were isolated.