Establishment of gene-knockout cell lines using the CRISPR/Cas9 system (CRISPR and Cas9 endonuclease system)

Cells were generated using a CRISPR-Cas9 strategy. The Cas9 lentivirus and lentiviral sgRNA vectors targeting EZH2 (sgEZH2) or scrambled control (sgNC) was purchased from Genechem (Shanghai, China). The sequence for the sgRNA targeting EZH2 was 5'-TGCGACTGAGACAGCTCAAG-3', and the negative-control sgRNA sequence was 5'-TTCTCCGAACGTGTCACGT-3', Cas9 lentiviral particles harboring a puromycin-resistance marker were transfected into SK-3rd cells. Lentiviral particles were added to cells at a multiplicity of infection of 10. After 24 h, the medium was replaced with full medium and cells were then passaged into selective medium with 2 μg/mL of puromycin until resistant Cas9-expressing cell (SK-3rd-cas9) colonies formed and antibiotic-induced cell death ceased. Then, the SK-3rd-cas9 cells were transfected with lentiviral vectors expressing both GFP and sgRNA targeting EZH2 (SK-3rd/sgEZH2). A scrambled control vector served as the negative control (SK-3rd/sgNC). After transfection, cells were incubated for 72 h. GFP-positive cells were isolated using limiting dilution and formed a single colony. The knockout efficiency was confirmed by Western blotting.

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