Site-specific gene correction of the β^s mutation in the HBB gene. (A) A scheme of stepwise gene correction of 1 mutant β^s allele (shown as 3 exons, 2 introns, and flanking sequences), first by HR-mediated gene targeting and followed by Cre-mediated excision. The gene-targeting donor BD2 vector with 2 homology arms (5.9-kb left arm and 2-kb right arm indicated by X) introduces an HR template for T-to-A replacement in the β^s allele and a loxP-flanked drug-selection cassette PGK-Hyg to be inserted into the HBB intron 1. The flanking counter-selection HSV-TK gene (in the form of a TK.GFP fusion) driven by the EF1α promoter (outside the right HR homology arm) is used to reduce the frequency of Hyg resistant clones because of BD2 vector random integration that also allows HSV-TK expression. For validated HR clones, Cre-mediated excision removes only the PGK-Hyg selection gene cassette and leaves 1 copy of the loxP DNA in the middle of HBB intron 1 of the corrected allele, generating “cre” clones with 1 corrected allele β^A (Corrected^ΔloxP). We used 2 PCR primers (red arrows) for initial screening of TI indicative of correct HR. (B) The initial results of gene targeting in 293T cells that were transfected with the 1-μg BD2 donor alone (lane 1) or with HBB-ZFNs at increasing amounts (0.25 μg in lane 2, 1 μg in lane 3, and 2 μg in lane 4). The 3′-TI event (2.5-kb PCR product) was detected when ZFNs were present. Untransfected 293T cells in lane 5 are negative. (C) Similar results of in selected clones after gene targeting in S1 iPSCs and Hyg and GVC selection. Four clones (c36, c64, c68, and c70) showed a positive PCR product. Positive control (p.c.) was DNA from targeted 293T cells (lane 3) in panel B. (D) Southern blot analysis surrounding the HBB locus in the parental S1 and selected targeted iPSC clones. A 3′-probe downstream of 3′-homology arm (top line) is used with genomic DNA digestion by PmeI (P) and EcoRV (E) enzymes to confirm the presence of targeted allele (with EcoRV site inside TI) shown as a green arrow, and the original HBB allele in a red arrow. (E) A Hyg probe is used with genomic DNA digestion by XbaI (X) and SpeI (S) enzymes to confirm the targeted allele (with Hyg insertion, green arrow) or to identify random integration events such as event in clone 65. (F) PCR screening for clones with successful Cre excision, using the 2 primers shown in red. The absence of the Hyg-containing DNA in clones such as cre16 and cre19 indicates the excision of the PGK-Hyg cassette. (G-H) Southern blot analyses of iPSC clones before and after Cre excision. (G) Results with the 3′-probe after P and E digestion as shown in panel D. Red arrows indicated 4.3-kb fragments from β^s allele in S1 and c36 iPSC clones and β^A allele in cre clones (4, 16, and 19). (H) Southern blots with Hyg probe after X and S digestion as shown in panel E. The S1 and 3 cre clones are free of the Hyg gene, which was found in c36 clone as expected (green arrow).