SCREENING AND PROTEIN-LEVEL VALIDATION OF SINGLE-CELL–DERIVED COA8 KNOCKOUT CLONES

Cytochrome c oxidase assembly factor 8 (COA8) is a protein involved in the assembly and function of mitochondrial complex IV (CIV).Complex IV is the final electron acceptor in the mitochondrial respiratory chain, transferring electrons from cyt c to molecular oxygen forming water and pumping protons. Pathological variants in COA8 are associated with isolated CIV deficiency and cavitating leukoencephalopathy, which is the loss of brain white matter. The aim of this study was to generate COA8 knockout human cellular models using an oligodendrocyte-derived cell line. MO3.13 oligodendrocyte-like cells were edited using CRISPR/Cas9 in order to disrupt the COA8 gene and Single-cell clones were isolated through clonal dilution and then expanded for protein extraction. The clones were analysed by SDS-PAGE and Western Blot/immunodetection of a CIV structural subunit (MTCO2). Then, a PCR of the possible KOs was performed in order to visualise indels and further Sanger sequencing. After SDS/WB screening of 107 single-cell derived clones, six clones were found to be possible KOs and were further analysed. Undoubtedly, the generation of MO3.13 COA8 KO cells is a step forward in the understanding of the patient's phenotype.