靶向小鼠Rbmy基因的CRISPR/Cas9系统高效切割位点筛选及其表达载体构建

Screening of the highly efficient cutting sites in the CRISPR/Cas9 system targeting the Rbmy gene of mouse and construction of the expression vector of the gene

性反转是指动物表现的性别特征与其应有的性别相反的现象。性反转小鼠可作为动物模型研究哺乳动物性别分化机制、性别控制技术以及研究人类性别连锁疾病发病机理和防治方法。已有研究证明,在胚胎期敲除雄性小鼠的Y染色体将获得XO基因型的性反转小鼠。本研究设计了6个不同的sgRNA,使共能介导Cas9蛋白作用于小鼠Y染色体上的多拷贝基因-Rbmy(RNA-binding motif gene),通过比较不同的sgRNA介导Cas9对靶序列的切割效率,最终筛选出一条能够高效介导Cas9靶向Rbmy基因的sgRNA6,该sgRNA6在体外切割效率达到100%,细胞内切割突变效率为15.4%,利用该sgRNA6构建了靶向Rbmy基因的CRISPR/Cas9系统表达载体。利用该载体转染小鼠雄性睾丸间质瘤细胞,成功获得了XO基因型的单细胞克隆团。本研究所构建的靶向Rbmy基因的CRISPR/Cas9系统表达载体可用于小鼠胚胎注射,为获得Y染色体敲除的XO型性反转小鼠模型提供基础。

Sexual reversal is a phenomenon in which the animals exhibit the opposite gender characteristics.Sexually reversible mice can be used as animal models to study the mechanisms of mammalian sex differentiation,sex control techniques,and the pathogenesis of and methods of preventing human sex-related diseases.Studies have shown that deletion of the Y chromosome at the embryonic stage result in sexually reversible mice with an XO genotype.In this study,six sgRNAs targeting a multi-copy gene,Rbmy,on the mouse Y chromosome,were designed.By comparing the cleavage efficiency of different sgRNA-mediated Cas9 for the target sequence,the sgRNA6 showing the highest efficiency was selected,whose the cleavage efficiency in vitro was 100%,and the cleavage mutation efficiency in cells was 15.4%.A sgRNA6-based CRISPR/Cas9 system expression vector targeting the Rbmy gene was constructed.The vector was transfected into the Leydig tumor cells of male mice,and a single cell colony with an XO genotype was successfully obtained.The Rbmy-targeting CRISPR/Cas9 system expression vector constructed in this study might be used to generate sexually reversed XO mice without the Y chromosome by embryo injection.