The mitochondrial Oxidative Phosphorylation (OXPHOS) system is the main site of ATP generation in most cell types and is encoded by more than 90 genes from both the nuclear and mitochondrial genomes. Of the OXPHOS enzymes, mitochondrial complex I is the largest (~1 MDa) composed of 44 subunits of dual genetic control and also the least well understood. This is mainly because the model organism Saccharomyces cerevisiae lack complex I and so many molecular and genetic tools used to study the other respiratory complexes cannot be applied to complex I biogenesis. This complex assembles via a number of intermediates and has been shown to require a number of chaperones known as assembly factors. These proteins guide the proper assembly of the intermediates into the active enzyme complex but are not required for the enzymatic activity of complex I. While the importance of assembly factors in complex I biogenesis has been demonstrated, the mechanism by which these assembly factors work remains elusive. Many patients diagnosed with mitochondrial disease and complex I deficiency have been identified with mutations in complex I assembly factor genes, however patient fibroblasts used in analysis have many drawbacks including their poor growth rate and transfection efficiency and the lack of isogenic control cells.

 This work aims to use the genome editing technology of transcription activator-like effector nucleases (TALENs) to introduce disruptions to the genes encoding complex I assembly factors in the HEK293T cell line to produce targeted knockouts. A cell line harbouring disruption to the FOXRED1 gene has been produced and has shown a reduction in the levels of complex I. This work aims to further investigate the function of these proteins at the molecular level to elucidate the role in complex I biogenesis and mitochondrial disease.