基于CRISPR/Cas9系统和增强ssODN重组建立tau-V337M小鼠模型

Development of a tau-V337M mouse model using CRISPR/Cas9 system and enhanced ssODN-mediated recombination

利用CRISPR/Cas9基因编辑技术建立tau-V337M突变的阿尔茨海默病(Alzheimer’s disease,AD)小鼠模型。通过设计和体外合成单向导RNAs(single guide RNAs,sgRNA)及单链寡核苷酸(single-stranded oligonucleotides,ssODN),将sgRNA、Cas9蛋白、ssODN注射到小鼠受精卵内,利用DNA切割和重组产生突变。为了提高重组效率,又在注射时添加Rad51蛋白。使用自然交配的雌鼠作为受体,将2细胞期的编辑胚胎进行单侧输卵管移植。研究发现通过添加Rad51蛋白可以获得较高的突变效率,在F0小鼠中获得了tau-V337M小鼠并进行扩繁,F0代tau-V337M小鼠可以将突变遗传给F1代。综上所述,本研究利用Cas9、ssODN和Rad51成功建立了首个tau-V337M基因位点突变的小鼠模型,为AD的研究和点突变模型制作提供了模型和方法基础。

The generation of a tau-V337M point mutation mouse model using gene editing technology can provide an animal model with fast disease progression and more severe symptoms,which facilitate the study of pathogenesis and treatment of Alzheimer’s disease(AD).In this study,single guide RNAs(sgRNA)and single-stranded oligonucleotides(ssODN)were designed and synthesized in vitro.The mixture of sgRNA,Cas9 protein and ssODN was microinjected into the zygotes of C57BL/6J mice.After DNA cutting and recombination,the site homologous to human 337 valine(GTG)in exon 11 was mutated into methionine(ATG).In order to improve the efficiency of recombination,a Rad51 protein was added.The female mice mated with the nonvasectomy male mice were used as the surrogates.Subsequently,the 2-cell stage gene edited embryos were transferred into the unilateral oviduct,and the F0 tau-V337M mutation mice were obtained.Higher mutation efficiency could be obtained by adding Rad51 protein.The F0 tau-V337M point mutation mice can pass the mutation on to the F1 generation mice.In conclusion,this study successfully established the first tau-V337M mutation mouse by using Cas9,ssODN and Rad51.These results provide a new method for developing AD mice model which can be used in further research on the pathogenesis and treatment of AD.