RESUMEN
Mitochondria are organelles present in the cytoplasm of eukaryotic cells; they play a key role in adenosine triphosphate (ATP) synthesis and oxidative phosphorylation. Mitochondria have their own DNA, mitochondrial DNA (mtDNA), keeping the function of the mitochondria. Mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters is and considered essential in mtDNA replication and transcription. More recently, TFAM has been shown to play a central role in the maintenance and regulation of mitochondrial copy number, inflammatory response, expression regulation, and mitochondrial genome activity. Gene editing tools such as the CRISPR-Cas 9 technique, TALENs, and other gene editing tools have been used to investigate the role of TFAM in mitochondrial mechanics and biogenesis as well as its correlation to mitochondrial disorders. Thus this chapter brings a summary of mitochondria function, dysfunction, the importance of TFAM in the maintenance of mitochondria, and state of the art of gene editing tools involving TFAM and mtDNA.
Asunto(s)
Edición Génica , Enfermedades Mitocondriales , Humanos , Dosificación de Gen , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/terapia , Enfermedades Mitocondriales/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The mitochondrial transcription factor A (TFAM) is considered a key factor in mitochondrial DNA (mtDNA) copy number. Given that the regulation of active copies of mtDNA is still not fully understood, we investigated the effects of CRISPR-Cas9 gene editing of TFAM in human embryonic kidney (HEK) 293T cells on mtDNA copy number. The aim of this study was to generate a new in vitro model by CRISPR-Cas9 system by editing the TFAM locus in HEK293T cells. Among the resulting single-cell clones, seven had high mutation rates (67-96%) and showed a decrease in mtDNA copy number compared to control. Cell staining with Mitotracker Red showed a reduction in fluorescence in the edited cells compared to the non-edited cells. Our findings suggest that the mtDNA copy number is directly related to TFAM control and its disruption results in interference with mitochondrial stability and maintenance.