QTRT1 Knockout in Breast Cancer Cells

The highly aggressive breast cancer cell line human MCF7 cells were cultured in a 6-well tissue culture plate in antibiotic-free MCF7 cell growth medium (Eagle’s minimum essential medium with 10% FBS, 10 µg/mL bovine insulin, and 10 nM β-estradiol; Invitrogen, Carlsbad, CA, USA) to 70%–80% confluence. Cells were transfected with 2 µg of QTRT1 Double Nickase Plasmid (sc-413456-NIC), 10 µL LTX Lipofectamine, and 2.5 µL PLUS Reagent (Invitrogen) per well (manufacturer’s protocol). The plasmid encodes a D10A mutated Cas9 nuclease and a unique, target-specific 20-nt guide RNA (gRNA), which has greater specificity of gene knockout than the CRISPR/Cas9 KO plasmid counterpart. QTRT1 Double Nickase Plasmid-derived puromycin resistance gene was used for positive selection of transfected cells. Cells were selected with 1 µg/mL puromycin for 5 days when further cell death was not observed. After selection, cells were collected and serial diluted onto four 96-well plates. Single cells were expanded to obtain individual clones for further study. The culture of MDA-MB-231 cells was as described before [53 (link)], and transfection and selection were as described above.