Objective: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.
Methods: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.
Results: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.
Conclusion: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.
题目: 双sgRNA介导的miR-155基因敲除对FLT3-ITD+AML细胞系的药物敏感性的影响.
目的: 在CRISPR/Cas9基因编辑系统中引入两条不同结合位点的sgRNA共转染FLT3-ITD+AML细胞系MV411获得miR-155基因敲除的MV411细胞，比较单sgRNA转染与双sgRNA转染法进行的miR-155基因敲除的效率及对细胞相关表型的影响.
方法: 以慢病毒为载体分别构建单转染与双转染的基因敲除细胞，PCR检测基因编辑效率。实时荧光定量PCR法检测miR-155-5p的表达水平。CCK-8法检测细胞增殖能力，并计算细胞对阿霉素与奎扎替尼的药物敏感性。Annexin V-APC/7-AAD双染色检测经阿霉素与奎扎替尼诱导的细胞凋亡水平.
结果: CRISPR/Cas9可对miR-155的编码基因进行有效编辑，双sgRNA转染细胞可见明显DNA剪接条带。与单sgRNA转染相比，双sgRNA转染中miR-155-5p的表达水平更低，对阿霉素与奎扎替尼具有更低的IC50，更高的药物诱导的细胞凋亡率.
结论: 双sgRNA转染对miR-155的基因表达的抑制率要高于单sgRNA转染。由双转染基因敲除所引起的细胞抑制增殖、逆转耐药、诱导凋亡等表型更为显著。与单一sgRNA转染相比，双转染sgRNA是一种高效的基因编辑方案.
Keywords: CRISPR/Cas9; FLT3-ITD; adriamycin; miR-155; quizartinib.