iPSCs were pretreated with 10 μM rho kinase inhibitor for 4 hours, and then 2 × 106 cells were electroporated using the Neon system with the Cas9/single-guide RNA ribonucleoprotein complex and the donor plasmid (synthesized by GeneArt). The donor plasmid contained three FLAG tags followed by a self-cleaving peptide (P2A) and a hygromycin resistance (HygroR). The 3xFLAG-P2A-HygroR cassette was flanked by BAZ1B-specific homology arms (5′ HA and 3′ HA) to promote homologous recombination and then subcloned into a bacterial backbone (Fig. 3A).

After 48 hours, iPSC medium was supplemented with hygromycin B (50 μg/μl), and selection medium was maintained for 15 days. Fifteen to 20 clones per iPSC line were then subjected to PCR to (i) evaluate the presence of the cassette and the insertion in the correct genomic locus and (ii) distinguish heterozygously tagged from homozygously tagged clones (fig. S3A). We could isolate a clone with a homozygous integration from the CTL, the atWBS, and the typical WBS but not from the 7dupASD line. In the 7dupASD clone, the FLAG tag was present in two of three copies, as shown by a digital PCR analysis (fig. S3B).

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