Analysis of APC-gp91 minigene plasmid ZFN-mediated HR targeting to the AAVS1 locus in X-CGD iPSCs. (A) Upper schematic of APC-gp91 minigene donor plasmid (pAAVS1-puro-CAG-gp91) and lower schematic of the PPP1R12C gene structure that includes exons 1, 2, and 3 flanking the AAVS1 locus that is within intron 1. Dotted lines indicate AAVS1 homology arms within the targeting donor plasmid. The puromycin-polyA promotorless cassette (SA-2A-Puro-pA) relies on a splice accepter plus 2A linker to use the PPP1R12C promoter for expression. The gp91^phox minigene cassette uses CAG (CMV early enhancer/chicken β-actin) promoter to express a codon-optimized gp91^phox cDNA-polyA. Restriction enzyme Sph1 (S) sites and 3′- and 5′-probes used for Southern blot are shown in both schematics. Not shown for the donor plasmid is a Sph1 restriction site 4.3 kb upstream of the 2 closely spaced Sph1 restriction sites within the schematic of pAAVS1-puro-CAG-gp91 plasmid. This upstream Sph1 site would not be present with a correctly targeted insert, but is often included with off-target random insertion of the donor plasmid. (B) Sph1 restriction Southern blot analysis of some of the APC-gp91 minigene targeted iXC9 (iXC9-APC-gp91) clones. 3′-probe (upper blot) detects wild-type allele (WT) 6.5 kb fragment in all clones except no. 20 (in which both alleles are targeted) and the 3′-targeted integration (TI) 6.0 kb fragment. 5′-probe (lower blot) detects WT 6.5 kb fragment in all but clone 20, the 5′ TI 3.8 kb fragment in all clones, and additional fragment(s) generated by random insertion in some clones (note the commonly seen 4.3 kb fragment derived entirely from donor plasmid sequence that is indicative of random insertion, and the 5.8 kb random insertion fragment seen in clone 20). From this particular analysis, clones indicated in red containing no random insert and a single-copy APC-gp91 insert in AAVS1 (nos. 9, 21, 23, 25, 26) were chosen for NHEJ screening (Table 3). (C) Flow cytometry gp91^phox transgene expression by iXC9-APC-gp91 c21 (right panel) and lack of gp91^phox expression by parental X-CGD iPSC clone iXC9 (left panel) (y-axis side scatter; x-axis fluorescent anti-gp91^phox). (D) iXC9-APC-gp91 c21 normal karyotype.