CRISPR Genome Editing in Mouse and Human Stem Cells

129/Sv × C57BL/6 ES cells were cultured on feeder cells using standard ES cell culture conditions. Cells were transfected with px330 expressing Cas9, mCherry, and sgRNA using Lipofectamine 3000 Reagent (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours after transfection, mCherry-positive ES cells were sorted into 96 wells using BD FACS AriaII for further culturing. After 7 days of culturing, the colonies were picked up and expanded for further analysis.
For cell treatment with drugs, the ATM inhibitor KU-55933 (number S1092, Selleckchem) was used at 20 μM. Cells were transfected with plasmids (pX330-mCherry-Ssty2-A and B) and mCherry-positive mouse ES cells were sorted by FACS 12 h after transfection. DNA-FISH analysis was performed 24 or 48 h later.
Human iPSCs were purchased from ATCC (ATCC® ACS-1003™) and cultured on irradiated mouse embryonic fibroblast (iMEFs) feeder layers in serum-free N2B27-LCDM medium as described previously [44 (link)]. For transfection, cells were dissociated using TrypLE, replated in iMEF-coated 12-well plates, and transfected in suspension with gRNAs, Cas9, and EGFP or mCherry plasmid using Lipofectamine 3000. Twenty-four hours after transfection, EGFP⁺/mCherry⁺ cells were sorted and used for DNA-FISH analysis at 7 days post-transfection.