A Novel Method for Detecting Activity of Mitochondria-targeted Nuclease

doi:10.13560/j.cnki.biotech.bull.1985.2015.12.012

Abstract

Abstract: 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.

Key words: mitochondria-targeted nuclease, transgenic cell line, targeted modification, CRISPR/Cas9, real-time fluorescence quantitative PCR

Wei Di, Gao Jing, Chi Zhenfen, Zhang Guirong, Nie Lingyun. A Novel Method for Detecting Activity of Mitochondria-targeted Nuclease[J]. Biotechnology Bulletin, 2015, 31(12): 81-90.

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