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

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.

A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication

“…The inability to culture cells with complete TFAM ablation [ 8 , 21 , 22 , 23 , 24 ] has impeded a more comprehensive understanding of this protein’s function in mtDNA replication, and the limited available evidence is derived predominantly from observations made in cells that co-express both wild type (wt) and altered forms of TFAM [ 8 , 25 ]. In these experiments, co-expression of the wt protein confounds interpretation and makes impossible unambiguous attribution of a phenotype to a specific TFAM mutation.…”

An Update on the Application of CRISPR Technology in Clinical Practice

“…Initially, to study mitochondrial diseases, cell lines and animal models of the disease were required, which were developed by genome-editing techniques such as CRISPR/Cas9 (e.g. NSUN2 knocked-out HEK293T cell line [ 149 – 151 ], YARS2 knocked-out HeLa cell line [ 152 ], and yars2 −/− zebrafish, nsun2 −/− mice [ 150 ] model). At the same time, the CRISPR technique is used in the development of Genomic-Wide Screening Libraries to identify essential genes in different pathways such as oxidative phosphorylation [ 153 ], ATP-modulating [ 154 ], cell death [ 155 ], metabolic resistance [ 156 ], adenine nucleotide translocator—ANT functions [ 157 ], or knock-out screen to identify how mitochondrial stress is relayed to ATF4 [ 158 ].…”