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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 n t检验。n 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 n in vitro.n 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 n t-test.n 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 n in vivo experiments.n