Direct evidence of CRISPR-Cas9-mediated mitochondrial genome editing

Pathogenic mitochondrial DNA(mtDNA)mutations can cause a variety of human diseases.The recent development of genome-editing technologies to manipulate mtDNA,such as mitochondria-targeted DNA nucleases and base editors,offer a promising way for curing mitochondrial diseases caused by mtDNA mutations.The CRISPR-Cas9 system is a widely used tool for genome editing;however,its application in mtDNA editing is still under debate.In this study,we developed a mito-Cas9 system by adding the mitochondria-targeted sequences and 30 untranslated region of nuclear-encoded mitochondrial genes upstream and downstream of the Cas9 gene,respectively.