小鼠及猕猴胚胎MECP2基因T158M单碱基突变体系的建立

T158M Single Base Editing of MECP2 Gene in Murine and Rhesus Monekey’s Embryos

目的:利用单碱基编辑技术定点修饰MECP2基因T158M位点,获得单一定点突变类型的啮齿类及非人灵长类胚胎,为建立模拟临床上Rett综合征的疾病动物模型奠定基础。方法:利用单碱基编辑技术构建3对T158M-sgRNA质粒,并分别与BE系统(BE3或BE4max)质粒共转染293T细胞筛选高效的sgRNA,通过显微注射将体外转录的mRNA以不同的浓度组合注射到ICR小鼠和猕猴受精卵中,检测小鼠和猕猴胚胎发育率和编辑效率,评估BE3和BE4max的工作效率及最优浓度组合。结果:小鼠胚胎上BE4max(ng/μl)∶sgRNA(ng/μl)=100∶50浓度组合时,小鼠胚胎发育率和编辑效率最佳,同时该浓度组合在猕猴胚胎上也获得了有效编辑效率。结论:成功在小鼠及猕猴胚胎上实现了MECP2基因T158M位点上C→T的转变,为后期建立T158M突变的RTT动物模型建立了稳定的胚胎编辑体系。

Objective:The T158M site of MECP2 gene was modified by using single-base editing technique to obtain single site-directed mutant rodent and non-human primate embryos,which lays a foundation for the establishment of an animal disease model simulating Rett syndrome in clinical practice.Methods:Three pairs of T158M-sgRNA plasmids were constructed using single-base editing technique,and co-transfected 293T cells with BE system(BE3 or BE4max)plasmids to screen highly-efficient sgRNAs.In vitro transcribed mRNAs were injected at different concentrations by microinjection to ICR mice and macaque fertilized eggs to measure the embryo development rate and editing efficiency of mouse and macaque and to evaluate the working efficiency of BE3 and BE4max and optimal concentration combination.Results:The mouse embryo development rate and editing efficiency are best when the concentration ratio of BE4max(ng/μl)∶sgRNA(ng/μl)on mouse embryos is 100∶50,and this concentration combination also shows effective editing efficiency on rhesus monkey embryo.Conclusion:The transition of C→T at position T158M of the MECP2 gene were successfully achieved in mouse and macaques embryos,establishing a stable embryo editing system for the later establishment of a T158M mutant RTT animal model.