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- Title
HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation.
- Authors
de Oliveira, Vanessa Cristina; Santos Roballo, Kelly Cristine; Mariano Junior, Clésio Gomes; Santos, Sarah Ingrid Pinto; Bressan, Fabiana Fernandes; Chiaratti, Marcos Roberto; Tucker, Elena J.; Davis, Erica E.; Concordet, Jean-Paul; Ambrósio, Carlos Eduardo
- Abstract
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.
- Subjects
CRISPRS; MITOCHONDRIAL DNA; GENOME editing; MITOCHONDRIA; STAINS &; staining (Microscopy); TRANSCRIPTION factors
- Publication
Life (2075-1729), 2022, Vol 12, Issue 1, p22
- ISSN
2075-1729
- Publication type
Article
- DOI
10.3390/life12010022