靶向小鼠Star基因的CRISPR/Cas9系统构建与打靶效力评价  

Construction of CRISPR/Cas9 system targeting mouse Star gene and evaluation of targeting efficacy

作　　者：韦妙灵 韦柳青 杨惠元 牧苏婉 李夏 李勇[1] WEI Miaoling;WEI Liuqing;YANG Huiyuan;MU Suwan;LI Xia;LI Yong(Intelligent Medicine and Biotechnology,Guilin Medical University,Guilin 541199,China;Key Laboratory of Brain and Cognitive Neuroscience,Guilin Medical University,Guilin 541199,China)

机构地区：[1]桂林医学院智能医学与生物技术学院,桂林541199 [2]桂林医学院广西脑与认知神经科学重点实验室,桂林541199

出　　处：《华夏医学》2024年第5期29-35,共7页Acta Medicinae Sinica

基　　金：国家自然科学地区科学基金项目(32260166);广西科技计划青年创新人才科研专项项目(2019AC20357);国家级大学生创新训练项目(202210601046,202310601047)。

摘　　要：目的利用CRISPR/Cas9系统对小鼠Star基因进行精确编辑,构建模拟人类STAR基因突变的细胞模型。方法选择中国先天性类脂质性肾上腺皮质增生症(CLAH)患者的热点突变区域,并设计相应的单导向RNA(sgRNA)。构建表达sgRNA的重组质粒,将其与慢病毒包装质粒共同转染至HEK-293T细胞并制备慢病毒颗粒以感染小鼠TCMK细胞。采用嘌呤霉素进行病毒感染后阳性细胞的筛选,提取阳性细胞的基因组DNA,通过聚合酶链式反应(PCR)扩增目标位点的基因组DNA片段,并利用TA克隆和Sanger测序技术,对编辑位点与效率进行统计分析。结果成功设计了有效的sgRNA序列,并利用CRISPR/Cas9系统成功构建了编辑小鼠Star基因的细胞模型。结论本研究通过精确编辑小鼠Star基因,成功构建了模拟人类STAR基因突变的CRISPR/Cas9系统,为深入研究CLAH的致病机制及探索潜在治疗方法提供有力工具,具有重要的科学意义和应用价值。Objective Using the CRISPR/Cas9 system to accurately edit the Star gene of mice,to simulate the mutation of the human STAR gene,and to construct an efficient gene editing model.Methods The hotspot mutation region of Chinese congenital lipoid adrenal hyperplasia(CLAH)patients was selected and the corresponding single directed RNA(sgRNA)was designed.A recombinant plasmid expressing sgRNA was constructed.HEK-293T cells were co-transfected with ecombinant plasmid expressing sgRNA and lentiviral packaging plasmid.Lentiviral particles were prepared,and mouse TCMK cells was infected.Puromycin was used to screen positive cells after viral infection,the genomic DNA of positive cells was extracted,the genomic DNA fragments of the target site were amplified through polymerase chain reaction(PCR).The editing site and efficiency were statistically analyzed by TA cloning and Sanger sequencing technology.Results The effective sgRNA sequences was successfully designed and a cell model for editing mouse Star genes using the CRISPR/Cas9 system was constructed.Conclusion In this study a CRISPR/Cas9 system that mimics human STAR gene mutations by accurately editing the mouse Star gene is successfully constructed.This provides a powerful tool for further studying the pathogenic mechanism of CLAH and exploring potential therapeutic methods and has important scientific significance and application value.

关 键 词：基因编辑 CRISPR/Cas9 单导向RNA 小鼠Star基因 先天性类脂质性肾上腺皮质增生症 

分 类 号：R394.3[医药卫生—医学遗传学]