Programmable aging function-related mitochondrial DNA 5-methylcytosine (m5C) modification with a TALE-directed methyltransferase

Mitochondrial epigenetic editing offers a potential strategy for modulating disease-causing mitochondrial genes while leaving the underlying DNA sequence unchanged. In this study, we present MEE, a mitochondrial epigenetic editor consisting of mitochondrion-targeted TALE modules fused to Dnmt3A and Dnmt3L methyltransferases. MEE efficiently directed site-specific 5mC methylation within cellular mitochondrial DNA with low detectable off-target activity under the tested conditions. MEE-mediated methylation at the C12191 (H) site exceeded 53% and was associated with an approximately 95% reduction in steady-state MT-ND5 mRNA levels in human cells. Notably, MEE increased methylation at the aging-associated C11168 (H) site by 11.76% in vivo, leading to reduced MT-ND4 expression in the targeted brain region and altered plasma levels of t-Tau and NFL in mice. These findings demonstrate that MEE provides a tool for precise epigenetic engineering of mitochondrial DNA, enabling experimental interrogation of specific 5mC modifications and exploration of the functional roles of mitochondrial DNA methylation in aging-associated diseases.