Investigation of reduced expression of the gene-targeted allele. (A) RT-PCR showed the same PGK-Hyg transgene was expressed at very low level in gene-targeted c36-iPSCs (lane 3) and c36-EryB (lane 4) compared with another iPSC line (FPHR, where the PGK-Hyg cassette was targeted into the actively expressed PIG-A gene,³  lane 1). Nontargeted S1-EryB cells were used as negative control (lane 2). (B) Sequencing of 3.5-kb genomic region of HBB locus in SCD iPSC clones. The 3.5-kb genomic region includes an ∼ 1.1-kb promoter, all the HBB exons and introns, and an ∼ 0.5-kb downstream sequence that were part of BD2 targeting donor (black lines/shapes/names) and also contains an ∼ 200-bp 3′-enhancer sequence (gray lines/shapes/names) downstream of the right homology arm. DNA sequencing of both alleles in early (p24) or late (p56) passage of S1 revealed uniform β^S mutation in exon 1 and wild-type GATA site in 3′-enhancer (underlined with complementary strand sequence underneath). However, in c36, cre4, or cre16, sequencing of mixed alleles showed heterozygous nucleotides (N), including β^A in exon 1, a G-to-T polymorphism near the 3′-end of the right homology arm, and an A-to-G mutation in GATA site all linked on gene-targeted allele.