利用CRISPR/Cas9系统构建稳定敲除anxa6基因的Caco-2细胞株

Knockout of human anxa6 gene in Caco-2 cells by CRISPR/Cas9 system

摘要:

【目的】利用CRISPR/Cas9系统构建稳定敲除anxa6基因的Caco-2细胞株，为研究大肠杆菌O157：H7效应蛋白EspF与宿主膜联蛋白A6（ANXA6）相互作用及其致病机制奠定基础。【方法】根据CRISPR/Cas9靶向原理设计并合成3个特异性识别anxa6基因的向导RNA （single guide RNA，sgRNA），基于LentiCRISPRv2载体构建LentiCRISPRv2-sgRNA重组质粒，转入293T细胞中，制备sgRNA-Cas9慢病毒，将慢病毒感染Caco-2细胞，经嘌呤霉素筛选阳性细胞，有限稀释法分离培养单克隆细胞，提取单克隆细胞基因组DNA，并对敲除位点附近的DNA片段进行PCR扩增，测序并进行脱靶效应评估；免疫印记法检测ANXA6蛋白表达情况，细胞计数试剂盒8（cell counting kit 8，CCK8）试剂盒检测细胞增殖能力，免疫荧光法检测细胞紧密连接分布情况。【结果】Western blotting及序列测序表明anxa6基因敲除单克隆细胞构建成功；脱靶效应评估结果显示预测的10个脱靶位点均无脱靶现象；基因敲除对细胞增殖能力无明显影响；免疫荧光结果显示，Caco-2及Caco-2^anxa6-/-细胞紧密连接蛋白ZO-1均沿细胞膜连续分布，结构完整，转染EspF质粒后出现分布不完整、缺口等现象。【结论】成功构建anxa6基因稳定敲除的Caco-2细胞株，初步探索ANXA6蛋白在紧密连接分布中的作用，为进一步研究大肠杆菌O157：H7通过EspF-ANXA6互作介导宿主肠屏障损伤分子机制提供技术支撑。

Abstract:

[Objective] To study the interaction between Escherichia coli O157:H7 effector protein EspF and host ANXA6 protein and its pathogenic mechanism, we constructed a stable Caco-2 cell line with the knockout of anxa6 using CRISPR/Cas9 system.[Methods] We designed and synthesized three small guide RNA (sgRNA) which can specifically recognize anxa6 gene. We constructed Lenticrisprv2-sgRNA recombinant plasmid and transfected it into 293T cells to prepare sgRNA-Cas9 lentivirus. Then we infected Caco-2 cells with lentivirus, and applied puromycin to screen the positive cells. We isolated the monoclonal cells by limiting dilution and sequenced the cells to evaluate the knock-out of gene anxa6 and the off-target effect. Western blotting was employed to detect the expression level of ANXA6, cell counting kit 8 (CCK8) assay to determine cell proliferation, and immunofluorescence to detect the distribution of tight junction protein ZO-1. [Results] The anxa6 gene in Caco-2 cell line was knocked out, and no off-target effect in the 10 predicted sites was found. The knockout of anxa6 had no significant effect on cell proliferation. ZO-1 of Caco-2 and Caco-2^anxa6‒/‒ cells displayed continuous distribution along the cell membrane, with complete structure. After transfection with EspF plasmids, the distribution of tight junction was incomplete with clear gaps and crack-like appearance. [Conclusion] We successfully constructed the Caco-2 cell line with the knock-out of anxa6. The cell line was used to preliminarily explore the role of ANXA6 protein in distribution of tight junction protein. This study provides an effective tool for exploiting the molecular mechanism of O157:H7 in mediating intestinal barrier injury through EspF-ANXA6 interaction.