# Targeting of Human Mitochondrial DNA with Programmable pAgo Nuclease

## TL;DR

Scientists successfully delivered a bacterial enzyme into human mitochondria, reducing mitochondrial DNA copy number using RNA guidance.

## Contribution

First demonstration of a bacterial Argonaute enzyme functioning inside mitochondria for targeted mtDNA manipulation.

## Key findings

- AmAgo was successfully delivered to mitochondria using the Su9 targeting sequence.
- RNA-guided AmAgo cleavage reduced mtDNA copy number by approximately 3-fold in human cells.
- The study establishes a new class of programmable tools for mitochondrial genome manipulation.

## Abstract

What are the main findings?
Targeted delivery of the prokaryotic Argonaute AmAgo into human mitochondria was achieved using the Su9 mitochondrial targeting sequence, resulting in robust mitochondrial localization.RNA-guided cleavage by AmAgo at single-stranded D-loop and R-loop regions of mitochondrial DNA resulted in a reproducible approximately 3-fold reduction in mtDNA copy number in human cells.

Targeted delivery of the prokaryotic Argonaute AmAgo into human mitochondria was achieved using the Su9 mitochondrial targeting sequence, resulting in robust mitochondrial localization.

RNA-guided cleavage by AmAgo at single-stranded D-loop and R-loop regions of mitochondrial DNA resulted in a reproducible approximately 3-fold reduction in mtDNA copy number in human cells.

What are the implications of the main findings?
pAgo nucleases represent a promising tool for targeted reduction in mitochondrial DNA copy number.Compact, PAM-independent pAgo nucleases can function inside mitochondria, establishing a new class of programmable tools for mitochondrial genome manipulation.

pAgo nucleases represent a promising tool for targeted reduction in mitochondrial DNA copy number.

Compact, PAM-independent pAgo nucleases can function inside mitochondria, establishing a new class of programmable tools for mitochondrial genome manipulation.

Manipulating the mitochondrial genome remains a significant challenge in genetic engineering, primarily due to the mitochondrial double-membrane structure. While recent advances have expanded the genetic toolkit for nuclear and cytoplasmic targets, precise editing of mitochondrial DNA (mtDNA) has remained elusive. Here we report the first successful mitochondrial import of a catalytically active RNA-guided prokaryotic Argonaute protein from the mesophilic bacterium Alteromonas macleodii (AmAgo). By guiding 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 establish a foundational framework for further development of programmable systems for mitochondrial genome manipulation.