Genome editing of hiPSCs PGP1 iPSCs were acquired from Personal Genome Project15. Cells were maintained on Matrigel (BD Biosciences)-coated plates in mTeSR1 medium (Stemcell Technologies) at 37?°C and 5% CO2 in a humidified incubator. Cultures were passaged every 5–7 days with TrypLE Express (Invitrogen). Genome editing on iPSCs using PiggyBac-inserted Cas9 was described before16. Briefly, PiggyBac carrying inducible Cas9 was integrated into the genome aided by transposase to generate Cas9-PGP1 iPSCs. After establishing the cell line, we transfected the cells with gRNA constructs through nucleofection and added 1?µg?ml-1 doxycycline to the media to induce Cas9 expression. Single cells were expanded into clones and on-target effects were validated through Sanger sequencing following the previously described protocol Site-specific deep sequencing Cas9-PGP1 iPSCs were nucleofected using P3 Primary Cell 4D-Nucleofector X Kit (Lonza). A total of 5 × 105 cells were harvested using TrypLE Express (Invitrogen) and resuspended in 20?µl nucleofection mixture containing 16.4?µl of P3 Nucleofector solution, 3.6?µl supplement, 1?µg of gRNA construct (and 1?µg Cas9 construct for transient transfection of Cas9). The nucleofection reactions were then conducted using the CB150 program. Thereafter the cells were plated on Matrigel-coated plates in mTeSR1 medium supplemented with ROCK inhibitor (Calbiochem Y-27632) and 1?µg?ml-1 DOX. After 48?h the cells were harvested and the genomic DNA was extracted using ZyGEM prepGEM extraction kit. Primers used in the deep sequencing can be found in Supplementary Table 3. The libraries were purified with QIAquick PCR Purification Kit (Qiagen) and sequenced on the MiSeq platform. Cas9 activity was measured through indel detection and relative Cas9 cutting efficiencies were calculated by normalising the off-target cutting efficiency to on-target efficiency.