Mitochondrial genome microhomology-mediated editing by donor DNA delivery into mitochondria in human cells.

Mutations in the mitochondrial DNA (mtDNA) are associated with severe human diseases, lacking efficient therapies. Direct correction of mtDNA mutations may offer a cure for such diseases. We propose a novel strategy based on double-stranded DNA (dsDNA) oligonucleotide delivery into mitochondria and intrinsic microhomology-mediated end joining (MMEJ) for mtDNA editing. This strategy enables introduction of multiple predefined nucleotide changes in mtDNA. For this, the presence of MMEJ activity in the human mitochondrial lysates was confirmed. 49 bp DNA oligonucleotide duplexes, fused to an RNA hairpin previously identified as a mitochondrial import signal, were delivered into the mitochondria of cultured human cells. Delivery of these donor dsDNA molecules, homological to an ND4 site of mtDNA and bearing designed nucleotide changes, led to a low but statistically significant introduction of the designed nucleotide changes into mtDNA. Donor dsDNA delivery combined with the CRISPR/mito-AsCas12a system also resulted in a statistically significant number of an expected concomitant change of five nucleotides distributed across a 16-nucleotide ND4 site of the mitochondrial genome. The proposed strategy may become an efficient mtDNA editing tool suitable for the correction of near-homoplasmic mutations such as Leber Hereditary Optic Neuropathy (LHON)-associated mutations in the ND4 gene of mtDNA.