旨在建立一种简便检测线粒体DNA（mtDNA）核酸酶靶向剪切活性的方法。利用转基因技术，将一段含有两个靶向目标序列（T1、T2）的线粒体DNA序列随机整合到宿主基因组中，通过实时荧光定量PCR筛选单拷贝或低拷贝的单克隆转基因细胞株。将含有T1、T2的CRISPR（clustered regularly interspaced short palindromic repeats）/Cas9质粒分别瞬时转染到所选细胞株中，靶向剪切核基因组，在靶向目标序列处造成DNA双链断裂，引发非同源末端连接修复机制，引入插入或缺失突变。观察测序峰图，证明两个靶向目标序列T1、T2均有剪切效率，且T1高于T2。建立了一种高效快速检测线粒体核酸酶靶向剪切活性的新方法。%Mitochondrial DNA(mtDNA)mutation has been associated with human mitochondrial disease. The precise correction of mutated mtDNA is considered an important strategy of mitochondrial therapy. Due to the lack of the complete repair system of DNA damage, a convenient method for detecting activity of mitochondria-targeted nuclease needs to be innovated urgently. Using transgenic technology, a mitochondrial DNA containing two target sequences(T1 and T2)was integrated into host genome randomly. Single or low copy monoclonal transgenic cell lines were selected by real-time fluorescence quantitative PCR. The two CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9 plasmids, which contained T1 and T2 target sequences, were transiently transfected into selected copy monoclonal transgenic cell lines. Target DNA double-strand breaks were caused by CRISPR/Cas9, followed by DNA insertion and deletion mutation via non-homologous end joining repair pathway. The cutting activities of two target sequences were proved by DNA sequencing peaks diagram, it was proved that two target sequences of T1 and T2 had the cutting activity, furthermore, T1 was better than T2. A novel method for rapidly and efficiently detecting activity of mitochondria-targeted nuclease is established.
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