CRISPR/Cas9技术的应用性研究

The Application Research of CRISPR/Cas9

旨在探索CRISPR/Cas9技术的新应用,本研究通过在小鼠遗传操作(Genetic manipulation)中使用该技术,创建了一种在活体组织内研究基因功能的方法。通过胚胎电转、免疫组化和PCR分析等手段,结果发现,针对DCX基因进行编辑,再现了DCX下调导致神经元迁移障碍的表型,说明该技术能够用于小鼠遗传操作中下调基因表达;其次,该技术还成功应用于Pcdhα基因簇的编辑并研究了基因簇恒定区的功能;最后,通过基因型鉴定发现,胚胎电转中使用CRISPR/Cas9会导致目的基因的敲除、反转和重复事件的发生。综上所述,在小鼠胚胎电转中使用CRISPR/Cas9技术,能够成功编辑目的基因,从而实现活体组织内研究基因的在体功能。

To investigate new application of CRISPR/Cas9 technology,we established a method to study genes function in living organism by the application of CRISPR/Cas9 in genetic manipulation.By using in vivo electroporation,immunohistochemistry and PCR analyzing,we first validated the function of DCXand the results indicated that CRISPR/Cas9 could be used in down-regulated gene expression in mouse genetic manipulation.Then this method was also used to study the function of constant exons of Pcdhαcluster.To further clarify the mechanism that led to the phenotype of neuron migration defects by DCX,we designed experiments to identify the genotype of target genes.The results indicated that CRISPR/Cas9 could lead to deletion,inversion and duplication of target genes.In conclusion,CRISPR/Cas9 technology could be used in mouse genetic manipulation and edit the target genes efficiently,which might be an ideal method to research gene function in living organism.