Fig. 3: Crispr/cas9 deletion of the CDKN1B gene induces resistance to the anticancer effect of Prozac.
a Parental or crispr/cas9-treated Kelly cell lines were cultured in the presence or absence of Prozac for 3 weeks in colony formation assays. Colonies were counted after staining cells with crystal violet. Bars indicate mean values ± SEM (n = 4). Statistical significance between different test conditions was determined using Student t test. Probability values < 0.05 were considered significant. A pair of single-guide RNAs (sgRNAs) was used to create two double strand breaks (DSBs) upstream and downstream of the CDKN1B locus, in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. The two specific sgRNAs were designed using the BlueHeron Guide RNA Target Design Tool and CasOT software:27 upstream sgRNA 5’-CGGCGACCTTCGCGGTCCTC-3’ and downstream sgRNA 5’-CAAAGGGACGTTCACGGCGA-3’. The sgRNAs were cloned into the pSpCas9-2A-GFP (PX458) vector (Addgene) and transfected into Kelly cells using Lipofectamine 3000 (Thermo Fisher Scientific). 48 h after transfection, GFP-positive cells were sorted by FACS (S3e™ Cell Sorter, Bio-Rad Laboratories, Inc., California, USA) and were plated individually into 96-well plates by limiting dilution. Clonal cell lines were expanded and gene deletion was tested by PCR, using the following primers: Fw 5’-TAAGTGCCGCGTCTACTCCT-3' and Rv 5’-AGCTTTCGCTGCTTTCTCAG-3'. Gene deletion was confirmed by DNA sequencing (Macrogen Spain, Madrid, Spain). PCR amplification and DNA sequencing of the non-deleted allele were performed using the following primers: Fw 5’-TAAGTGCCGCGTCTACTCCT-3' and Rv 5’-ATACGCCGAAAAGCAAGCTA-3'. b Crispr/cas9 targeted Kelly clones 16 and 10 were subjected to western blot analysis with a p27Kip1 antibody. An actin antibody was used as loading control. c Ectopically overexpressed p27Kip1 induces growth arrest of SKNAS cells. Cells were seeded in 60-mm dishes at a density of 1 × 106 cells/dish and transfected with control (pcDNA3) or pcDNA3-p27 vectors using Lipofectamine 2000 (Invitrogen, 11668-019) following manufacturer’s instructions. 72 h after transfections, the cells were used for western blot analysis to verify expression of ectopic levels of p27Kip1 (left panel) or plated into a six-well plate at a density of 5000 cells/well and selected with 800 µg/ml G418. 3 weeks later, colonies were fixed with cold methanol, stained with 0.5% crystal violet and quantified (right panel). Bars indicate mean values ± SEM (n = 3).
