基于CRISPR/Cas9基因编辑技术的RIP1稳定敲除细胞模型的构建及功能研究

Construction and characterization of RIP1 stable knockout HaCaT cell line based on CRISPR/Cas9 system

目的利用CRISPR/Cas9技术构建RIP1稳定敲除的人永生化表皮细胞HaCaT细胞株,在此基础上对RIP1功能进行探究。方法针对GenBank中RIP1基因序列,设计靶向RIP1不同外显子的小指导RNA(sgRNA),并将其克隆至SpCas9-2A-Puro V2.0(PX459)载体中,获得PX459-sgRNA基因敲除载体。将上述载体转染HaCaT细胞并通过嘌罗霉素筛选获得阳性克隆,免疫印迹和DNA测序技术进一步鉴定RIP1敲除效果最佳的单克隆。通过CCK-8实验检测RIP1缺失对细胞增殖能力的影响。分别用TNF-α处理HaCaT^(WT)细胞和HaCaT^(R1P1KO)细胞,流式细胞仪检测敲除RIP1对TNF-α诱导细胞死亡的影响,进一步通过胱天蛋白酶(caspase)抑制剂Z-VAD-FMK判断细胞死亡类型。结果与结论利用CRISPR/Cas9系统成功构建RIP1完全敲除的细胞模型,功能分析发现敲除RIP1导致细胞增殖能力减慢;HaCaT^(R1P1KO)对TNF-α诱导的死亡畀常敏感,这种死亡能被Z-VAD-FMK显著抑制,证明为caspase依赖性细胞凋亡。该研究为探究RIP1在皮肤损伤中的作用奠定了基础。

Objective To establish a stable RIPl gene knockout cell line using CRISPR/Cas9 system in immortalized human epidermal cell line HaCaT,and to explore the function of RIP1 in HaCaT cells by this novel HaCaT（RIPIKO） cell line.Methods The small guide RNA（sgRNA） sequences targeting different exons of RIP1 designed according to sequence in GenBank were cloned into the SpCas9-2A-Puro V2.0 vectors to construct the recombinant PX459-sgRNA plasmid for RIP1 gene knockout.The HaCaT cells transfected with this recombinant plasmid were selected by puromycin to obtain the positive clone cells.Then,the monoclones with optimal gene knockout effect were further screened out by Western blotting and sequencing.CCK8 assay was perfonned to investigate the effect of RIPl knockout on proliferation of HaCaT cells.The TNF-α-induced cell death of HaCaT（WT） cells and HaCaT（RiPIKO） cells was detected by flow cytometry,and the type of cell death was further determined using the caspase inhibitor Z-VAD-FMK.Results and Conclusion The HaCaT（RIPIKO）￣ cell line was successfully constructed using CRISPR/Cas9 system.RIPl knockout significantly reduced the proliferation of HaCaT cells.Besides,the HaCaT（RIPIKO） cells were more vulnerable than HaCaT（WT） cells to the TNF-α-induced cell death,which was dramatically rescued by Z-VAD-FMK,indicating that the process is caspase-dependent apoptosis in HaCaT（RIPIKO） cells.This work may contribute to the study of the functions of RIP1 in skin lesions.