Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria.

Area of Science:

• Cell Biology
• Genetics
• Molecular Biology

Background:

• Mitochondrial DNA (mtDNA) mutations cause severe diseases, but engineering large-scale mtDNA deletions in human cells remains challenging.
• Previous advances allowed base substitutions and removal of mutated mtDNA, but not the creation of disease-relevant deletions.

Purpose of the Study:

• To develop a novel method for engineering specific, large-scale mtDNA deletions in human cells.
• To create and characterize a panel of isogenic cell lines with varying levels of mtDNA deletions.
• To investigate the cellular and molecular consequences of mtDNA deletions.

Main Methods:

• Co-expression of prokaryotic end-joining (EJ) machinery and targeted endonucleases (mito-EJ and mito-ScaI) to engineer mtDNA deletions.
• Generation of clonal cell lines with a ~3.5 kb mtDNA deletion across the full spectrum of heteroplasmy.
• Analysis of cellular phenotypes, including OXPHOS protein levels, metabolic function, and growth.
• Single-cell multiomic analysis to identify nuclear gene expression changes.

Main Results:

• Successfully generated a panel of human cell lines with specific mtDNA deletions and controlled heteroplasmy levels.
• Identified a critical heteroplasmy threshold of approximately 75% deleted genomes, beyond which OXPHOS depletion, metabolic disruption, and impaired growth occurred.
• Discovered two distinct patterns of nuclear gene deregulation in response to mtDNA deletions: one triggered at the threshold and another that progressively responds to increasing heteroplasmy.

Conclusions:

• The co-expression of mito-EJ and programmable nucleases is a powerful tool for modeling mtDNA deletion diseases.
• The established cell line panel provides a valuable resource for studying the impact of mtDNA deletions.
• This approach can guide the development of therapeutic strategies for mitochondrial myopathies and age-related diseases.