HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation
oleh: Vanessa Cristina de Oliveira, Kelly Cristine Santos Roballo, Clésio Gomes Mariano Junior, Sarah Ingrid Pinto Santos, Fabiana Fernandes Bressan, Marcos Roberto Chiaratti, Elena J. Tucker, Erica E. Davis, Jean-Paul Concordet, Carlos Eduardo Ambrósio
| Format: | Article |
|---|---|
| Diterbitkan: | MDPI AG 2021-12-01 |
Deskripsi
The mitochondrial transcription factor A (<i>TFAM</i>) 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 <i>TFAM</i> 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 <i>TFAM</i> 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 <i>TFAM</i> control and its disruption results in interference with mitochondrial stability and maintenance.