TALEN designs and constructions were based on a Golden Gate Assembly protocol with modifications to the vector backbone33. Donor DNA vectors with a loxP-flanked PGK-hygromycin cassette were cloned between 5′ and 3′ homology arms (Fig. 3a), which were amplified from genomic DNA of a healthy subject and a patient with the DISC14-bp mutation. For targeting, TALENs (4 μg DNA of each plasmid) and linearized donor vectors (10 μg DNA) were electroporated into individual iPS cells (1 × 106 to 2 × 106 cells pretreated with 5 μM ROCK inhibitor, Y-27632, Cellagentech) using Nucleofector 2b (Lonza; program A-023). Transfected cells were transferred onto a 6-well dish pre-plated with inactivated MEFs and supplemented with Y-27632 in standard iPS cell medium. Positive colonies were selected by 10mg ml−1 hygromycin B (Invitrogen) after 5 days of culture or until small colonies appeared. Resistant colonies were sub-cloned and expanded in 48-well plates. Over 200 clonal lines were screened. The loxP-flanked PGK-hygromycin cassette was removed by electroporation of a Cre recombinase expression vector (4 μg DNA). Specific integration, correct genetic editing and efficient removal of PGK-hygromycin cassette at each stage were verified by Sanger sequencing.
