Schematic diagram of NCF1 and its pseudogenes on chromosome 7 and the corrective donors used. (A) Positions of pseudogenes (NCF1B and NCF1C) relative to NCF1 are shown in the inset of chromosome locus 7q11.23. (B) Distinguishing sequences in normal NCF1 and pseudogenes include unique intronic SNPs, the normal GTGT (or mutant ΔGT) at start of exon 2 in NCF1 vs constitutive ΔGT in both NCF1B and NCF1C as depicted. We do not indicate in this figure that a portion of pseudogene or an entire pseudogene may replace a portion of or the entire NCF1 gene at its NCF1 locus. ZFN targets the start of exon 2, with the recognition sequence (upper case) and the spacer or cutting region (lower case) as shown. The ZFN targets the pseudogene sequence shown: CCCAGGTACATGTTCctggtgAAATGGCAGGAC. The capital letters represent the target sequence, and the lowercase letters denote the space or cutting site. Within the “Donor repair sequence,” the underlined base pair G was changed from a base pair A to avoid recutting but does not change the codon translation and is the codon-optimized choice. (C) Schematic representation of the correction donors used for minigene addition or exon 2 replacement with or without a puromycin (Puro) selection cassette flanked with loxP (A/B or C/D, respectively). The cytomegalovirus (CMV) promoter is used to express the puromycin resistance gene and the polyadenylation signal (pA) is indicated. Shown are left and right homology arms (LHA and RHA, respectively). The inverted terminal repeats (ITRs) in donors B, C, and D allow donor packaging in rAAV vector.