To construct the eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) gene knockout human breast cancer MCF-7 and ZR-75-1 cell lines using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated 9 (Cas9) gene editing technology.

We designed a sequence-specific guide RNA (sgRNA) that specifically targeted the 4EBP1 gene, and used the lentiCRISPR v2 plasmid as a backbone to construct a recombinant plasmid that expressed sgRNA and Cas9 protein. After sequencing and identification, the recombinant plasmid was co-transferred into HEK 293T cells with retroviral packaging plasmids pMD2.G and psPAX2 for lentiviral packaging, and viral supernatants at 24 h and 48 h were collected to infect human breast cancer MCF-7 and ZR-75-1 cells. MCF-7 and ZR-75-1 cells with deletion of 4EBP1 were screened out using puromycin, and verified by DNA sequencing and Western blot analysis. The MCF-7, ZR-75-1 wild-type and 4EBP1 knockout cells were transfected with pcDNA3-rluc-poli-IRESfluc plasmid. The effects of different mutants of 4EBP1 on cap-dependent translation were detected using a dual luciferase reporter gene assay and the cap-dependent translation level was expressed as the ratio of renilla luciferase to firefly luciferase (R/F ratio). The t-test was used to compare the R/F ratio between groups, and the LSD method was used for pairwise comparisons.

Western blot analysis showed that the expression levels of 4EBP1 protein were significantly reduced in MCF-7 and ZR-75-1 cells transfected with the three constructed lentiCRISPR v2-4EBP1-KO plasmids, indicating successful knockdown of 4EBP1 gene. DNA sequencing revealed a T-base insertion of sgRNA in the DNA sequence of MCF-7 cells, and a GC-base deletion of sgRNA in the DNA sequence of ZR-75-1 cells, demonstrating the success of gene editing in the cellular genomic DNA. The R/F ratios measured in wild-type and 4EBP1 knockout ZR-75-1 cells were 1.83±0.10 and 5.48±0.33, respectively, suggesting a significant difference between two groups (t=-18.338, P<0.001). The R/F ratios measured in wild-type and 4EBP1 knockout MCF-7 cells were 1.17±0.06 and 2.03±0.20, respectively, suggesting a significant difference between two groups (t=-7.167, P<0.001). The R/F ratios measured in ZR-75-1 cells with 4EBP1 knockout, and ZR-75-1 cells transfected with 4EBP1, plv-neo-4EBP1-37/46 and plv-neo-4EBP1-4A were 5.48±0.33, 3.83±0.10, 3.53±0.23 and 1.87±0.05, respectively, suggesting a significant difference between groups (F=151.995, P<0.001); the corresponding R/F ratios measured in MCF-7 cells were 2.03±0.200, 1.37±0.14, 1.90±0.19 and 1.20±0.17, respectively, suggesting a significant difference between groups (F=5.744, P=0.001).

The 4EBP1 knockout breast cancer cell lines can be used for the studies on the role of different 4EBP1 phosphorylation mutants on translation initiation.