4.6 Article

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

Journal

LIFE-BASEL
Volume 12, Issue 1, Pages -

Publisher

MDPI
DOI: 10.3390/life12010022

Keywords

CRISPR-Cas9; gene editing; HEK293T cells; mitochondrial DNA; TFAM

Funding

  1. CNPq [431508/2018-6]

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This study investigated the effects of CRISPR-Cas9 gene editing of TFAM in HEK293T cells on mtDNA copy number. The findings suggest that editing TFAM can decrease mtDNA copy number and interfere with mitochondrial stability and maintenance.
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.

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