Fig. 4

Biallelic modification of the APRT locus. a Schematic overview of the method for scarless engineering of APRT*J and control alleles. Homology arm overlap generates a 32 bp tandem µH (blue), with the patient mutation (c.407T > C, red) present unilaterally, and the Silent mutation (c.402A > T, blue) present bilaterally. A GFP reporter is included in the backbone to exclude cells with random donor integration by FACS. Gene targeting used CRISPR-Cas9 (yellow bolt, Supplementary Fig. 13). The remaining elements are as described in Fig. 2a. b Detailed schematic of APRT gene targeting and MMEJ resolution. The heterozygous SNP (rs8191489) is shown in orange. Additional labeling is consistent with Fig. 2b. c Southern blot analysis of select APRT hetero- and homozygously targeted clones using genomic (APRT-5’, top) and transgenic (mCherry, bottom) probes. Parental 1383D6 iPSCs are included as a control. “x” indicates one clone with aberrant banding. d Histograms of mCherry fluorescence intensities in select APRT targeted clones. Note that the bimodal distribution is correlated with genotype, and therefore CAG::mCh transgene copy-number. e APRT diploid genotypes of clones. Heterozygous genotypes were resolved using TIDE. Alleles marked as ‘APRT*J’ were also edited with the Silent mutation. f Southern blot analysis of select excised clones revealing restoration of the APRT locus (APRT-5’ probe, top) and removal of the cassette (mCherry probe, bottom). Parental 1383D6 and intermediate targeted iPSCs (from c) are included as controls. Genotypes (S, Silent only; A, APRT*J and Silent) are indicated above