Genome Editing with AsiSI Endonuclease

U20S were retrieved from ATCC and modified with a plasmid encoding for the restriction enzyme (pBABE-AsiSIER and pAID-AsiSIER)^{29 (link),30 (link)}. U2OS, DIvA (AsiSI-ER-U20S), and AID-DIvA (AID-AsiSI-ER-U20S) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogen) and either 1 µg/mL puromycin (DIvA cells) or 800 µg/mL G418 (AID-DIvA cells) at 37 °C under a humidified atmosphere with 5% CO₂. The cell lines were regularly checked for mycoplasma contamination. For AsiSI-dependent DSB induction, cells were treated with 300 nM 4OHT (Sigma, H7904) for 4 h. When indicated, 4OHT-treated cells were washed three times in pre-warmed PBS and further incubated with 500 µg/mL auxin (IAA) (Sigma; I5148) for the indicated time. For transcriptional inhibition, DRB (Sigma, 100 μM) or cordycepin (Sigma, 50 µM) was added to the medium 1 h prior to 4OHT (4 h) and auxin (2 h) treatments. Cells were arrested in G1 using a 48 h treatment with 40 μM lovastatin (Mevinolin from LKT Laboratories) and in G2 with a 24 h treatment with 40 μM Ro-3306 (CDK1 inhibitor, Calbiochem). For clonogenic assays in U20S cells, DSBs were induced either by increasing doses of etoposide (Sigma) for 16 h as indicated or by irradiation with a Cs137 source (Biobeam 8000).