CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome

• DOI: 10.1007/s11427-020-1819-8
• Publication Date: 2021-01-09T11:47:15Z
• Authors: Bang Wang; Xiujuan Lv; Yufei Wang; Zhibo Wang; Qi Liu; Bin Lu; Yong Liu; Feng Gu
• Keywords: Gene Editing; 0303 health sciences; 03 medical and health sciences; DNA Repair; INDEL Mutation; Genome, Human; Mutagenesis; Genome, Mitochondrial; Humans; CRISPR-Cas Systems; DNA, Mitochondrial; Mitochondria

Genetic manipulation of mitochondrial DNA (mtDNA) could be harnessed for deciphering the gene function of mitochondria; it also acts as a promising approach for the therapeutic correction of pathogenic mutation in mtDNA. However, there is still a lack of direct evidence showing the edited mutagenesis within human mtDNA by clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9). Here, using engineered CRISPR/Cas9, we observed numerous insertion/deletion (InDel) events at several mtDNA microhomologous regions, which were triggered specifically by double-strand break (DSB) lesions within mtDNA. InDel mutagenesis was significantly improved by sgRNA multiplexing and a DSB repair inhibitor, iniparib, demonstrating the evidence of rewiring DSB repair status to manipulate mtDNA using CRISPR/Cas9. These findings would provide novel insights into mtDNA mutagenesis and mitochondrial gene therapy for diseases involving pathogenic mtDNA.