Generation of induced pluripotent stem cells and gene correction

Skin fibroblasts were reprogrammed by nucleofection with pCXLE- hOct3/4 (RRID:Addgene_27076), pCXLE-hSK (RRID:Addgene_27078),using the Amaxa nucleofection kit for human dermal fibroblasts (Lonza, VPD-100) and program P-022 of the Nucleofector 2b (Lonza).

Nucleofected fibroblasts were plated in six-well plates coated with Matrigel (Corning)

in DMEM supplemented with 10% FBS (Gibco) and 1% GlutaMAX Supplement (Gibco).

The following day, the medium was changed to DMEM+/+ (DMEM with 10% FBS and 1% GlutaMAX Supplement and 1% Pen/Strep (Millipore)) supplemented with 2 ng/ml recombinant

basic human fibroblast growth factor (FGF2, Peprotech).

On day 3 or4 post nucleofection, the medium was changed to E8 medium com-

posed of DMEM F12 with HEPES (Gibco), 128 ng/ml ascorbic acid (Sigma

–Aldrich), 1x insulin-transferrin-selenium (Thermo Fisher

Scientific), 10 ng/mL FGF2 (Peprotech), 500 ng/ml heparin (Sigma-Aldrich), and 2 ng/ml TGFβ1 (Peprotech). E8 medium was supplemented with 100 μm sodium butyrate and 0.1% Pen/Strep.

Colonies started to appear from day 14 onward.

Induced pluripotent stem cells (iPSCs) were cultured on Vitronectin XF (StemCell Technologies) in E8 medium.

Gene correction for the L444P mutation was performed as previously described in (Schöndorf, D. C. et al., 2014)

One hour before nucleofection, 10μM Rockinhibitor was added to the iPSC medium. About 240 nM crRNA (IDT): Atto550-labeled tracrRNA (IDT) duplex was complexed with 124μM Cas9 to form the ribonucleoprotein complex (RNP complex).

iPSCs(1.6 × 10^6) were nucleofected with the RNP complex and 16μg of ssODN using 100μl of Ingenio nucleofection solution (Mirus) with program B-016 of Nucleofector 2b.

Following nucleofection, the cells were FACSsorted for Atto550-positive cells using a FACS Aria II with a 100-μmnozzle. After sorting, 1 × 10^4 cells were plated per 10-cm dish.

Colonies were picked and sequenced by Sanger sequencing.