CRISPR Editing of U2OS Cells

U2OS cells (a gift from Toni Cathomen, Freiburg) and U2OS.EGFP cells (containing a single integrated copy of an EGFP-PEST reporter gene)^{30 (link)} were cultured in Advanced DMEM supplemented with 10% HI FBS, 2 mM GlutaMax, and penicillin/streptomycin at 37°C with 5% CO₂. The growth media for U2OS.EGFP cells was additionally supplemented with 400 µg ml⁻¹ Geneticin. All cell culture reagents were obtained from Life Technologies. Cell line identity was validated by STR profiling (ATCC) and deep-sequencing, and cells were tested bi-weekly for mycoplasma contamination. Unless otherwise noted, cells were co-transfected with 750 ng of Cas9 plasmid and 250 ng of sgRNA plasmid. For negative control experiments, Cas9 plasmids were co-transfected with a U6-null plasmid. Nucleofections were performed using the DN-100 program on a Lonza 4-D Nucleofector with the SE Cell Line Kit according to the manufacturer’s protocol (Lonza). For T7E1 assays, GUIDE-seq experiments, and targeted deep sequencing, genomic DNA was extracted ~72 hours post-transfection using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter Genomics).