CRISPR/Cas9系统介导的p53基因突变促进树鼩骨骼肌卫星细胞的增殖活性

Mutation of p53 gene mediated by CRISPR/Cas9 system promoting proliferative activity of skeletal muscle satellite cells in tree shrews

目的:利用CRISPR/Cas9系统在树鼩骨骼肌卫星细胞中敲除p53基因并检测其增殖活性。方法:通过生物信息学方法对树鼩和15种哺乳动物的p53蛋白氨基酸序列进行同源性分析,根据树鼩基因组信息设计可编辑树鼩p53突变位点sgRNA,构建lenti CRISPR v2-sgp53基因编辑重组质粒,用293T细胞进行慢病毒包装,感染树鼩骨骼肌卫星细胞,嘌呤霉素筛选多克隆细胞并扩增;通过测序确定基因编辑效果。采用CCK8法检测细胞增殖活性,采用western blotting验证p53蛋白表达水平。结果:与人p53和小鼠p53氨基酸序列同源性(96.92%)相比,人p53和树鼩p53的氨基酸序列同源性(98.21%)更高。进化树分析显示,与小鼠相比,人与树鼩的亲缘关系更近。成功构建了3个靶向树鼩p53编辑的重组载体:lenti CRISPR v2-sgp53-g1、lenti CRISPR v2-sgp53-g2、lenti CRISPR v2-sgp53-g3,均成功在树鼩骨骼肌卫星细胞上实现p53基因编辑产生突变,导致p53功能丧失,p53的突变提高了树鼩骨骼肌卫星细胞的增殖能力。经药筛后的树鼩骨骼肌卫星细胞p53基因敲除成功,p53蛋白表达水平显著降低(P<0.01)。结论:本研究利用CRISPR/Cas9系统成功使树鼩骨骼肌卫星细胞p53基因突变、功能丧失,为后续树鼩p53基因敲除模型的建立提供了重要的分子生物学基础。

Objective:To knock out p53 gene in skeletal muscle satellite cells in tree shrews and detect its proliferative activity through utilization of the CRISPR/Cas9 system.Methods:The homology analysis of amino acid sequences of the p53 protein in tree shrews and 15 mammals was conducted using a bioinformatics approach.Based on the genomic information of tree shrews,a specific sgRNA targeting the p53 mutation site was designed.Subsequently,a lenti CRISPR v2-sgp53 gene editing recombinant plasmid was constructed and lentiviral packaging was performed with 293T cells to infect skeletal muscle satellite cells in tree shrews.Polyclonal cells were then screened and amplified using puromycin.The effectiveness of gene editing was determined by sequencing analysis.Cell proliferation activity was assessed through cell counting kit-8(CCK8) assay,and the expression level of p53 protein was verified by western blotting.Results:Compared to the amino acid sequence homology of human p53 and mouse p53(96.92%),the homology between human p53 and tree shrew p53(98.21%) was higher.Evolutionary tree analysis revealed a closer relationship between humans and tree shrews than mice.Three recombinant vectors targeting p53 editing in tree shrews were successfully constructed,namely lenti CRISPR v2-sgp53-g1,lenti CRISPR v2-sgp53-g2,and lenti CRISPR v2-sgp53-g3,and they all successfully achieved p53gene editing in skeletal muscle satellite cells in tree shrews to produce mutations,resulting in loss of p53 function.The mutation of p53 improved the proliferation of skeletal muscle satellite cells in tree shrews.The p53gene was knocked out successfully in skeletal muscle satellite cells in tree shrews,and the expression level of p53protein was significantly decreased(P<0.01).Conclusion:In this study,the CRISPR/Cas9 system is used to successfully mutate and disable p53 gene in skeletal muscle satellite cells in tree shrews,which provides an important molecular biological basis for the subsequent establishment of p53 gene knockout model in tree shrews.