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.
Highlights
TFAM is a key mitochondrial modulator with a proposed role in mitochondrial DNA transcription and replication, and is required to regulate mtDNA copy numbers [1,2]
Both gRNAs resulted in genome editing of the first exon of TFAM
Given the important role of mitochondria in cellular homeostasis, dysfunction of this organelle can lead to several common diseases [23]
Summary
TFAM is a key mitochondrial modulator with a proposed role in mitochondrial DNA (mtDNA) transcription and replication, and is required to regulate mtDNA copy numbers [1,2]. TFAM is an abundant protein, present in approximately 1000 molecules per mtDNA molecule in mammalian cells, which is enough to coat the entire mitochondrial genome [3]. It has been shown that TFAM is directly involved in mitochondrial genome repair by modulating the access of repair proteins to mtDNA [4,5]. Alterations in mtDNA, such as a decrease in quantity, can cause impaired energy production leading to several clinical disorders. The result of this dysfunction can cause damage to mtDNA compromising mitochondrial function [6,7].
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