Targeting of human mitochondrial DNA with programmable pAgo nuclease

Manipulating the mitochondrial genome remains a major challenge in genetic engineering, largely due to the mitochondrial double-membrane structure. While recent advances have expanded the genetic toolkit for nuclear and cytoplasmic targets, precise editing of mtDNA has remained elusive. Here we report the first successful mitochondrial import of a functional RNA-guided prokaryotic Argonaute protein from the mesophilic bacterium Alteromonas macleodii (AmAgo). By directing AmAgo to the single-stranded D- or R-loop region of mtDNA using synthetic RNA guides, we observed a nearly threefold reduction in mtDNA copy number in human cell lines. This proof of concept study demonstrates that a bacterial Argonaute can remain active within the mitochondrial environment and influence mtDNA levels. These findings lay the groundwork for further development of programmable systems for mitochondrial genome manipulation.