Specific elimination of m.8993T>G mitochondrial haplotype in NARP cybrid cells by CRISPR-Cas9 system

Mutations in mitochondrial DNA can cause a wide range of neuromuscular and neurodegenerative diseases in humans. The heteroplasmy level, coexistence of both wild-type and mutant mtDNA within a cell, determines the manifestation and severity of disease symptoms. Therefore, the development of strategies to shift heteroplasmy toward wild-type mtDNA is critical for advancing therapies for mitochondrial disorders. Mitochondrial localization of the modified CRISPR-Cas9 system components was analyzed using western blotting, immunocytochemical staining, confocal microscopy, and immunoelectron microscopy. To assess the heteroplasmy shift, cybrid cell lines carrying the clinically relevant m.8993T>G variant associated with Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome were used. The heteroplasmy level was evaluated by RFLP analysis of PCR-amplified mtDNA fragments encompassing the mutation site. We adapted the widely used CRISPR-Cas9 system, originally designed for editing nuclear DNA, to induce a heteroplasmy shift of the pathogenic m.8993T>G point mutation in human cybrid cells. We demonstrated that the modified components of the CRISPR-Cas9 system – the mitoCas9 nuclease fused to the mitochondrial targeting sequence from COX8A, and a single guide RNA containing a mitochondrial import determinant within the tetraloop – are effectively imported into the mitochondrial matrix. Transient transfection of the modified sgRNA into cybrid cells stably expressing mitoCas9 resulted in a detectable shift in heteroplasmy. Mitochondria-targeted CRISPR-SpCas9 system can selectively reduce the mutant m.8993T>G mtDNA load in human cybrid cells, achieving a reproducible shift in heteroplasmy.