构建人源化CYP4V2点突变小鼠的CRISPR/Cas9系统  

A humanized murine model of bietti crystalline dystrophy

作　　者：赵莹科 方源 钱丽玲 王伟 吴继红 ZHAO Yingke;FANG Yuan;QIAN Liling;WANG Wei;WU Jihong(Department of Ophthalmology,Eye&ENT Hospital,Fudan University,Shanghai 200031,China;Key Laboratory of Visual Impairment and Restoration(Fudan University),Shanghai 200031,China;NHC Key Laboratory of Myopia(Fudan University),Shanghai 200031,China;CAS center for excellence in brain science and intelligence technology,Shanghai 200031,China)

机构地区：[1]复旦大学附属眼耳鼻喉科医院眼科,上海200031 [2]上海市视觉损伤与重建重点实验室,上海200031 [3]国家卫生健康委员会近视眼重点实验室,上海200031 [4]中国科学院脑科学与智能技术卓越创新中心,上海200031

出　　处：《中国眼耳鼻喉科杂志》2022年第6期584-588,共5页Chinese Journal of Ophthalmology and Otorhinolaryngology

基　　金：上海市市级科技重大专项(2018SHZDZX05);上海市科委生物医药专项(21S11905900)。

摘　　要：目的应用CRISPR/Cas9构建携带人源化CYP4V2突变的小鼠模型,为遗传性视网膜变性提供与临床疾病病变特征相一致的模式动物。方法应用生物信息学方法,分析现有CYP4V2中我国眼遗传病患者常见突变热点,并根据其致病性以及序列比对,最终选定突变位点。按照tild-CRISPR的方法,构建供体质粒,以及Cas9系统。最后将供体、Cas9mRNA以及gRNAs注射到小鼠受精卵中,测序鉴定是否构建成功。结果通过gnomAD以及ClinVar数据库,筛选CYP4V2常见的热点突变,经过序列比对,最终选定CYP4V2 c.1091-2 A>G为后续构建模型的致病突变。最终,DNA测序支持构建完成的基因编辑鼠在CYP4V31091-2的位置发生了A>G的突变。mRNA水平不能确定是否发生可变剪切。结论我们利用CRISPR/Cas技术,成功制备了携带患者特异性遗传突变的小鼠模型,为以后深入探索CYP4V21091-2 A>G点突变的致病机制以及开发相应的基因治疗药物奠定了基础。Objective To establish a humanized knock-in mouse model which mimics the CYP4V2mutation identified in patients with(Bietti crystalline dystrophy, BCD). Methods We identified the most common CYP4V2 missense variant which was correlated with BCD in clinic. Then, we constructed the appropriate donor DNA carrying human mutation. By injecting the donor, Cas9mRNA, and gRNAs into the zygote, the mouse models were generated. The sequence was determined accordingly. Results The mutation of 1091-2A>G in CYP4V2 which can lead to the crystalline retinitis pigmentosa was determined firstly. Then the related components were successfully injected to the mice fertilized egg to generate mouse model. At DNA level, homologous replacement of the mouse CYP4V3 with human sequence carrying 1091-2 A>G mutation was achieved successfully. The mRNA levels cannot determine whether alternative splicing has occurred.Conclusions A BCD mouse model carrying CYP4V3(ortholog of human CYP4V2 in mice) 1091-2 A>G point mutations was generated successfully. It would be beneficial to exploring the pathogenic mechanism of BCD and developing corresponding gene therapy.

关 键 词：CRISPR/Cas9 人源化小鼠 CYP4V2基因 结晶样视网膜变性 

分 类 号：R774.1[医药卫生—眼科]