Nucleofection of iPSC:
Adherent cells were dissociated using Tryple (Gibco), centrifuged and resuspended in TeSR containing 10µM ROCK inhibitor, Y27632. 1×106 cells were resuspended in 100µl of Lonza® Nucleofection solution (according to manufacturer’s protocol). The following amounts of DNA were added; Cas9 2µg, gRNA 2 µg, PBHR 5µg, pmaxGFP 2µg, and/or PiggyBac Transposase 2.5µg in the required experimental combination. The samples were then nucleofected using the B-16 protocol on the device. After nucleofection, TeSR was added and cells transferred to a 6-well Matrigel-coated plate. After 24–48 hours of incubation, the nucleofected iPSC were split onto 10cm MEF plates at single-cell density for colony screening or harvested for genomic DNA analysis.
PiggyBac Transposase Nucleofection (excision):
Excision of the integrated selection cassette from corrected clones was done by overexpressing piggyBac transposase in clonal iPSC lines expressing puromycin resistance and sensitivity to ganciclovir. 4µg of various piggyBac transposase vectors (System Biosciences) were nucleofected into 1×106 iPSC using the Amaxa Nucleofector Device as described above, followed by selection with 3µg/ml ganciclovir to eliminate unexcised cells.