构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究  

Construction of inducible CRISPR/Cas9 system for studying gene function in mouse

作　　者：赵艳娜 邱荣 沈南[1] 唐元家[1] ZHAO Yan-na;QIU Rong;SHEN Nan;TANG Yuan-jia(Shanghai Institute of Rheumatology,Department of Rheumatology,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200127,China;Shanghai Institute of Nutrition and Health,Chinese Academy of Sciences,Shanghai 200031,China)

机构地区：[1]上海交通大学医学院附属仁济医院风湿病科,上海市风湿病学研究所,上海200127 [2]中国科学院上海营养与健康研究所,上海200031

出　　处：《上海交通大学学报（医学版）》2021年第3期297-301,共5页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81871287,31630021);上海交通大学医学院高水平地方高校创新团队(SSMU-ZDCX20180100)。

摘　　要：目的·结合Dox诱导型单链导向RNA(single guide RNA,sgRNA)表达载体和Cas9转基因小鼠,构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究。方法·根据四环素诱导表达系统原理,基因合成U6-TetO-sgRNA和EF1α-T2A-Puro-BFP-T2ATetR片段。通过同源重组将2个片段组装进反转录病毒载体骨架,获得Dox诱导型sgRNA反转录病毒载体。为了验证系统有效性,分离Cas9转基因小鼠骨髓细胞并诱导其向巨噬细胞方向分化。设计对照(non-targeting control,NC)组和实验组(靶向F4/80)的sgRNA,利用反转录病毒感染细胞,分化条件设置添加Dox组(Dox^(+))和不添加Dox组(Dox^(-))。通过流式细胞术和T7核酸内切酶Ⅰ(T7 endonucleaseⅠ,T7EⅠ)实验检测基因敲除效果。结果·①成功构建Dox诱导型sgRNA反转录病毒表达载体和Cas9转基因小鼠。②流式结果显示,在NC Dox^(-)组、NCDox^(+)组和F4/80 Dox^(-)组中,几乎无F4/80阴性细胞群体;而在F4/80 Dox^(+)组中,F4/80阴性细胞群体高达50%。③T7EⅠ结果显示,在F4/80 Dox^(-)组中,DNA条带完整,而在F4/80 Dox^(+)组中发生基因突变,DNA条带被切开。结论·结合Dox诱导型sgRNA表达载体和Cas9转基因小鼠,成功构建诱导型CRISPR/Cas9系统。利用该系统成功在小鼠免疫细胞中实现可诱导性基因敲除。Objective·To construct inducible CRISPR/Cas9 system for studying gene function in mouse immune cells,combining Dox^(-)inducible single guide RNA(sgRNA)expression vector with Cas9 transgenic mice.Methods·U6-TetO-sgRNA and EF1α-T2A-Puro-BFP-T2A-TetR fragments were obtained by gene synthesis.The two synthetic fragments were assembled into the retroviral vector backbone by using homologous recombination.sgRNA targeting protein coding region of F4/80 and non-targeting control(NC)were designed.Bone marrow cells were isolated from Cas9 transgenic mice and transfected with retrovirus expressing sgRNA.The experimental conditions were divided into Dox^(-)added group(Dox^(+))and non Dox^(-)added group(Dox^(-)).The knockout effect was tested by flow cytometry and T7 endonucleaseⅠ(T7EⅠ)experiments.Results·①Dox^(-)inducible sgRNA retroviral vector and Cas9 transgenic mice were successfully constructed.②The result of flow cytometry showed that F4/80 was only knocked out in the F4/80 Dox^(+)population,but not in NC Dox^(-),NC Dox^(+)and F4/80 Dox^(-)populations.③T7EⅠresults showed that the DNA was cut into two bands in the F4/80 Dox^(+)group,while the DNA band was intact in the F4/80 Dox^(-)group.Conclusion·An inducible CRISPR/Cas9 system combining Dox^(-)inducible sgRNA retroviral vector with Cas9 transgenic mice are successfully constructed.With this system,inducible gene knockout in mouse immune cells are successfully achieved.

关 键 词：诱导型CRISPR/Cas9系统 Dox诱导型sgRNA表达载体 造血干细胞 基因编辑 巨噬细胞 

分 类 号：R394.4[医药卫生—医学遗传学]