CRISPR/Cas9介导下构建FN1敲除的DLD1细胞系及初步功能学研究

DLD1 cell line with FN1 knock-out mediated by CRISPR/Cas9 and its preliminary functional study

[目的]利用CRISPR/Cas9技术构建FN1基因敲除的人结直肠腺癌DLD1细胞系,并初步探讨FN1基因敲除对DLD1细胞增殖、迁移的影响。[方法]根据CRISPR/Cas9技术原理,设计针对FN1基因的crRNA,体外混合crRNA、tracrRNA、Cas9酶,形成核糖核蛋白(即RNP复合物)后转染至DLD1细胞,实现基因编辑。采用二维梯度稀释法挑取单克隆细胞系,使用测序及蛋白质印迹技术(Western Blotting)检测FN1基因敲除情况,通过CCK8、Transwell及划痕实验检测FN1敲除对DLD1细胞增殖、迁移的影响。[结果]测序结果表明,8号单克隆细胞系在靶点附近缺失1 366个碱基,造成FN1编码基因的移码突变,蛋白翻译提前终止。与未编辑细胞相比,DLD1FN1-KO细胞的增殖及迁移能力明显降低。[结论]成功构建DLD1FN1-KO细胞系。初步证实,与未编辑细胞相比,DLD1FN1-KO细胞在第5d增殖水平减慢约40%(P <0.001)、迁移水平减慢约27%(P <0.001)。

[Objective] To investigate the effect of fibronectin( FN1) on proliferation and migration of DLD1 cells by using FN1 knockout DLD1 cells generated with CRISPR/Cas9 genomic editing technology. [Method] Specific FN1 targeting crRNA was designed according to the principle of CRISPR/Cas9 technology. crRNA: tracrRNA duplex and Cas9 enzymes were mixed in vitro to form a functional transfection complex( RNP complex). Monoclonal cell lines were obtained through two-dimensional gradient dilution method,and FN1 gene knockout was detected by DNA sequencing as well as Western Blotting. Moreover,CCK8,transwell and wound healing assays were applied to evaluate the proliferation and migration ability of DLD1FN1-KO cells.[Result]CRISPR/Cas9 mediated genomic editing of FN1 gene generated a 1 366 bases deletion in No. 8 monoclonal cell line,leading to the frameshift mutation of the FN1 gene and early termination of protein translation. Compared with unedited cells,cell proliferation and migration abilities were crippled in DLD1FN1-KO cells.[Conclusion]DLD1FN1-KO cell line was constructed expectedly. It was preliminarily confirmed that the proliferation level of DLD1FN1-KO cells was reduced by about 40%( P <0. 001) on the 5th day,and the migration level was decreased by about 27%( P < 0. 001) compared with that of unedited cells.