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目的:应用腺相关病毒(AAV)-规律间隔成簇短回文重复序列及其相关核酸酶9(CRISPR/Cas9)系统体外实验研究基因治疗威尔森氏症(WD)。方法:选用2月龄雄性Toxic milk (TX)小鼠作WD模型，设计小向导RNA(sgRNA)并构建含有CRISPR元件的AAV载体质粒命名为pAAV-sgRNA1、2、3，制备AAV，病毒滴度达1～4×10n 13 GC/ml；感染WD肝细胞，72 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，筛选出其中活性最高者sgRNA1，构建相应的同源修复模板(HT)质粒命名为pAAV-HT，应用AAV-sgRNA1和AAV-HT共感染WD肝细胞，测序和T7E1酶切检测模板修复效率；用HT特异性引物行PCR，验证HT掺入。组间比较采用Student’s n t检验。n 结果:Sanger测序结果显示，sgRNA1在体外有最高的编辑效率[(20.2±2.3)%]，与sgRNA2、3比较[(3.1±1.3)%、(14.6±2.0)%]，差异有统计学意义(n P<0.05)。进一步用HT进行基因修正，能检出特异性修正后的ATP7B基因；二代测序测序显示修复率为(7.9±2.7)%。n 结论:AAV-CRISPR系统在体外可以达到较高编辑效率和修复效率。“,”Objective:To study gene therapy for Wilson′s disease (WD) with adeno-associated virus (AAV)-clustered regularly interspaced short palindromic repeats (CRISPR) system n in vitro.n Methods:Three small-guide RNAs (sgRNAs) were designed and AAV particles containing CRISPR-sgRNA1, 2, 3 or W/O homologous template (HT) were transduced into Toxic milk (TX) mouse hepatocytes, respectively. Gene editing efficiency and HT repair efficiency were evaluated by sequencing, and repair template-specific primers were used for PCR to verify HT incorporation.Results:Sanger sequencing data showed that the editing efficiency of sgRNA1 reached (20.2±2.3)%, which was significantly higher than others (n P<0.05). When AAV-HT treatment was combined, the corrected ATP7B gene band could be amplified by PCR, and NGS showed that repair efficiency was (7.9±2.7)%.n Conclusion:AAV transduction can achieve pretty high editing efficiency. We selected sgRNA1 and verified its editing efficiency and the repair efficiency of HT, providing a theoretical and experimental basis for further n in vivo experiments.n