Site-specific homologous recombination (HR) mediated by CRISPR/Cas9 in β-thalassemia iPSCs. (A) PCR analyses using two pairs of primers, P4/P5 and P1/P2, each pair with one in the piggyBac and the other in the HBB outside the targeting construct to detect homologous recombination. (B) Bacterial artificial chromosome (BAC) containing the piggyBac cassette inserted in the TTAA site located in intron 1 of the wild-type HBB used as a positive control; (P) parental line, (M) marker. (B) Homologous recombination confirmed by Southern blot analysis. Southern blot analysis after HindIII digestion of genomic DNA from puromycin-resistant clones using a 5′ genomic probe outside the targeting construct (left) and a Neo probe inside the piggyBac (right). Clone 75 shows the correct 13-kb band marked by an asterisk (*), detected by both the 5′ globin gene and Neo probes, indicating site-specific homologous recombination at the globin locus. The 8-kb bands are from the endogenous HBB. Other bands seen with the Neo probe are due to random integrations.