Generation of stable knockout cell lines using CRISPR-cas9

Guide RNAs against human AhR, CDKN1B, and TP53 genes in one vector CRIPSR-cas9 lentiCRISPRv2 plasmid have been described (31) and were purchased from GeneScript (Piscataway, NJ). Stable AhR, CDKN1B (p27) and TP53 (p53) knockout lines were generated using CRISPR-cas9 system as previously described in detail (72). Briefly, for each gRNA, viral particles were generated by co-transfecting CRISPR-cas9 sgRNA plasmid with packaging plasmids psPAX2 and pMD2.G (Addgene, Watertown, MA) into HEK293T cells using lipofectamine 2000 (ThermoFisher, Waltham, MA). The viral particles were collected twice on the next two days for a total 4 ml of supernatant containing viral particles, frozen in −80°C overnight, filtered through 0.2 μm filter, divided into 0.5 ml aliquots and kept in −80°C. H460 or H69AR cells were transduced by reverse transduction with the viral particles in RPMI 1640 medium supplemented with 1% FBS and 10 μM protamine sulfate. Infected cells were selected for using 0.5 μg/ml puromycin for one week. Monoclonal cell lines were generated by limited dilution in 96-well plates. After 10 days in culture, cells were checked under a microscope and wells containing only one clone were selected for further expansion. Knockout phenotype was confirmed by Western Blot. Guide RNAs against AhR have been described in our previous study (55); guide RNAs targeting human p27 and p53 genes: p27 gRNA-1 ATT GCT CCG CTA ACC CCG TC; p27 gRNA-2 GGG TTA GCG GAG CAA TGC GC; p27 gRNA-3 TTC CCC AAA TGC CGG TTC TG; p53 gRNA-1 CCG GTT CAT GCC CAT GC; p53 gRNA-2 CGC TAT CTG AGC GCT CA; p53 gRNA-3 CCC CGG ACG ATA TTG AAC AA.