应用规律间隔成簇短回文重复序列及其相关核酸酶9系统核糖核蛋白复合物技术基因修正威尔森氏症的体外研究  

In vitro study of gene therapy for Wilson’s disease with clustered regularly interspaced short palindromic repeats ribonucleoprotein method

作　　者：史啸立 杨人杰 李相成 俞悦 Shi Xiaoli;Yang Renjie;Li Xiangcheng;Yu Yue(Department of Hepatobiliary Surgery,the First Affiliated Hospital of Nanjing Medical University,Nanjing,Key Laboratory on Living Donor Liver Transplantation,National Health and Family Planning Commission,Nanjing 210029,China)

机构地区：[1]南京医科大学第一附属医院肝胆中心/肝移植中心国家卫健委活体肝移植重点实验室中国医学科学院活体肝移植重点实验室,210029

出　　处：《中华实验外科杂志》2020年第12期2226-2228,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81570565)。

摘　　要：目的应用规律间隔成簇短回文重复序列及其相关核酸酶9系统(CRISPR/Cas9)体外实验研究修正威尔森氏症(WD)基因。方法选用来源于DL近交系小鼠的Toxic milk小鼠(购自美国Jackson Laboratory公司)作WD模型,设计3个CRISPR来源的RNA(crRNA)-crRNA001/002/003,应用CRISPR/Cas9核糖核蛋白复合物(RNP)方法转染WD肝细胞,48 h后提取基因组DNA,聚合酶链反应(PCR)后测序检测基因编辑效率,筛选出效率最高者crRNA001,并设计相应的单链寡核苷酸(ssODN)修复模板;用crRNA001和ssODN共转染肝细胞,模板特异性引物行PCR,验证ssODN掺入。组间比较采用Student’S t检验。结果Sanger测序结果显示,crRNA001的编辑效率为(57.8±5.1)%。用ssODN共转染细胞后,模板特异性引物行PCR,能扩增出修正后的铜离子转运ATP酶β肽(ATP7B)基因条带;二代测序结果显示,ATP7B基因修复效率为(7.45±2.54)%。结论CRISPR/Cas9 RNP转染能达到较高编辑效率,相应的ssODN可以高效修复缺陷ATP7B基因,为进一步的体内实验提供理论和实验基础。Objective To study gene therapy for Wilson’s disease with Clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 system in vitro.Methods Three crRNAs,W/O homologous template,the single-stranded oligo DNA(ssODN),were transfected into Toxic milk(TX)mouse hepatocytes using CRISPR/Cas9 ribonucleoprotein(RNP)method,respectively.Gene editing efficiency and ssODN repair efficiency were verified by sequencing,and repair template-specific primers were used for PCR to verify ssODN incorporation.Data are expressed as mean±standard deviation of independent samples.Comparisons between two groups were performed by unpaired Student’s t-test.Results Sanger sequencing showed that the editing efficiency of crRNA001 reached(57.8±5.1)%.The corrected ATP7B gene band can be amplified by PCR using specific primers,and NGS sequencing results showed that the ATP7B gene repair efficiency reached(7.45±2.54)%.Conclusion Pretty high editing efficiency can be achieved by RNP transfection.We selected crRNA001 and verified its editing efficiency and the repair efficiency of ssODN,providing a theoretical and experimental basis for further in vivo experiments.

关 键 词：威尔森氏症 成簇的规律间隔的短回文重复序列 基因治疗 铜离子转运ATP酶β肽 核糖核蛋白体 

分 类 号：R742.4[医药卫生—神经病学与精神病学]