Targeting of Human Mitochondrial DNA with Programmable pAgo Nuclease

Area of Science:

• Mitochondrial biology
• Genetic engineering
• Molecular biology

Background:

• Mitochondrial genome manipulation is difficult due to the organelle's structure.
• Current genetic tools are limited for targeting mitochondrial DNA (mtDNA).

Purpose of the Study:

• To demonstrate the first successful mitochondrial import and activity of a bacterial Argonaute protein.
• To establish a programmable system for manipulating the mitochondrial genome.

Main Methods:

• Imported a catalytically active Argonaute protein from *Alteromonas macleodii* (AmAgo) into human cell mitochondria.
• Utilized synthetic RNA guides to direct AmAgo to mtDNA regions (D-loop or R-loop).

Main Results:

• Observed a nearly threefold reduction in mtDNA copy number in human cell lines.
• Confirmed that the bacterial Argonaute protein remains active within the mitochondrial environment.

Conclusions:

• Bacterial Argonaute proteins can be functionally imported into mitochondria.
• This work provides a foundation for developing targeted mitochondrial genome editing technologies.