HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation

The mitochondrial transcription factor A ( ) 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 in human embryonic kidney (HEK) 293T cells o...

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Published in:Life (Basel, Switzerland) Switzerland), 2021-12, Vol.12 (1), p.22
Main 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
Format: Article
Language:English
Subjects:
Cloning
Control stability
Copy number
CRISPR
CRISPR-Cas9
Disruption
Efficiency
Fluorescence
gene editing
Gene regulation
Genetic modification
Genomes
HEK293T cells
Mitochondrial DNA
Mutation
Mutation rates
Ovaries
Proteins
TFAM
Variance analysis
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Summary:The mitochondrial transcription factor A ( ) 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 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 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 control and its disruption results in interference with mitochondrial stability and maintenance.
ISSN:2075-1729
2075-1729
DOI:10.3390/life12010022