CRISPR-Cas9 Mediated αTAT1 Knockout in MDA-MB-231 Cells

MDA-MB-231 was kindly provided by Dr. JS Nam (Gwangju Institute of Science and Technology [GIST], Korea). αTAT1 KO MDA-MB-231 cells were generated using the CRISPR-Cas9 system as described in a recent paper (Ko et al., 2021 (link)). Briefly, Lentiviral particles expressing a guide sequence (5’-CATGAGTCTGTGCAACGCCA-3’) targeting Atat1 were produced by transfection of HEK293T cells with lentiCRISPR v2 plasmid, psPAX2, and pMD2.G using polyethylenimine for 48 h (Oh et al., 2017 (link)) MDA-MB-231 cells transduced with the lentivirus were selected with puromycin (1 μg/ml) for 2 weeks. Hs578t was purchased from the Korean Cell Line Bank. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) with high glucose, 10% fetal bovine serum, 100 unit/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B (Gibco) in a humidified incubator at 37°C and 5% CO₂. All cell lines were confirmed not to be infected with mycoplasma using e-Myco VALiD Mycoplasma PCR Detection kit (iNtRON, Korea).