Cell culture and stable cell line generation with CRISPR/Cas9

Human ovarian cancer cell lines A2780, C13, ES-2, HO8910, OVCAR-3, and SKOV3 were purchased the American Type Culture Collection (ATCC, Manassas, VA, USA). Human normal ovarian epithelial cell line IOSE was supplied by Heng-Yu Fan, Zhejiang University [43]. Cells were grown in DMEM (Gibco | Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco | Thermo Fisher Scientific) and 1% penicillin-streptomycin (Gibco | Thermo Fisher Scientific) at 37 ℃ in humidified atmosphere composed of 5% CO₂ and 95% air (standard culture conditions).

DCAF13-deficient cells were established using CRISPR/Cas9 technology. The guide RNA sequences used for targeting human DCAF13 were: human DCAF13–1: 5’- AGCGGGACAGCAGTGAGCCC-3’; human DCAF13–2: 5’-GATGTGGATTACTCTCCCAC-3’. The guide RNA sequences were inserted into LentiCRISPRv2 (addgene, #98290). The 293T cells were seeded into 6-well plates at a density of 100,000 cells/well. After 24 h, the medium was replaced with fresh DMEM supplemented with 10% FBS. To make lentivirus, 293T cells/well were cotransfected with a recombinant lentiviral vector LentiCRISPRv2 (400 ng) with the packaging plasmids pMD2.G (200 ng) and psPAX2 (200 ng) by using poly-jet (SignaGen, Rockville, MD, USA). After 48 h transfection, the 293T cell supernatant containing virus was filtered through a 0.45–µm hydrophilic polyvinylidene difluoride (PVDF) membrane (Millex-HV, Millipore, Burlington, MA, USA) to remove cell debris. The filtered 2 mL lentivirus were transfected into 4T1, 4T07, and MCF-7 cells by using polybrene (final concentration 8 ug/mL). Twenty-four hours after transfection, 1–2 ug/mL puromycin (Gibco) was continuously added to the cells for 3 days to sort transfected cells. Viable cells were sorted into 96-well plates with a single cell in each well. Colonies were grown and clones were screened by Western blot analysis using antibodies against DCAF13.