利用规律性重复短回文序列簇/相关基因9(CRISPR/Cas9)编辑技术构建真核翻译起始因子4E结合蛋白1(4EBP1)基因敲除的人乳腺癌MCF-7、ZR-75-1细胞系。

设计能特异性针对4EBP1基因的特异性向导RNA(sgRNA)，以lentiCRISPR v2质粒为骨架构建能表达sgRNA和Cas9蛋白的重组质粒。测序鉴定后将重组质粒与逆转录病毒包装质粒pMD2.G、psPAX2共同转入HEK 293T细胞进行慢病毒包装，收集病毒上清感染人乳腺癌MCF-7、ZR-75-1细胞。利用嘌呤霉素筛选4EBP1表达缺失的MCF-7、ZR-75-1细胞，DNA测序和Western blot验证。在MCF-7、ZR-75-1野生型与4EBP1基因敲除的细胞中转入pcDNA3-rluc-poli-IRESfluc质粒，并用双荧光素酶报告基因实验检测4EBP1、4EBP1不同突变体对帽子依赖翻译的影响，以海肾荧光素酶与萤火虫荧光素酶比值(R/F比值)来表示帽子依赖的翻译水平。R/F比值的组间比较采用t检验，两两比较采用LSD法。

Western blot结果显示构建的3种lentiCRISPR v2-4EBP1-KO重组质粒转染MCF-7、ZR-75-1细胞后，4EBP1蛋白的表达水平均明显降低，说明4EBP1基因敲除成功。DNA测序发现MCF-7细胞的DNA序列中sgRNA出现了T碱基的插入突变，在ZR-75-1细胞DNA序列中sgRNA出现了GC碱基的缺失突变，证明细胞基因组DNA中的基因编辑成功。野生型和4EBP1基因敲除的ZR-75-1细胞中测得的R/F比值分别为1.83±0.10和5.48±0.33，组间比较差异有统计学意义(t＝－18.338，P<0.001)。野生型和4EBP1基因敲除的MCF-7细胞中测得的R/F比值分别为1.17±0.06和2.03±0.20，组间比较差异有统计学意义(t＝－7.167，P<0.001)。4EBP1基因敲除、敲除后分别转染4EBP1、plv-neo-4EBP1-37/46和plv-neo-4EBP1-4A的4种ZR-75-1细胞中测得的R/F比值分别为5.48±0.33、3.83±0.10、3.53±0.23和1.87±0.05，组间比较差异有统计学意义(F＝151.995，P<0.001)；4种MCF-7细胞中测得的R/F比值分别为2.03±0.200、1.37±0.14、1.90±0.19和1.20±0.17，组间比较差异有统计学意义(F＝5.744，P＝0.001)。

4EBP1基因敲除乳腺癌细胞系可用于后续不同4EBP1磷酸化突变体对翻译起始作用的进一步研究。

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.