HEK293T cells with TFAM disruption by CRISPR-Cas9 as a model for mitochondrial regulation

Vanessa Cristina de Oliveira*, Kelly Cristine Santos Roballo, Clésio Gomes Mariano Junior, Sarah Ingrid Pinto Santos, Fabiana Fernandes Bressan, Marcos Roberto Chiaratti, Elena J. Tucker, Erica E. Davis, Jean Paul Concordet, Carlos Eduardo Ambrósio

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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.

Original languageEnglish (US)
Article number22
JournalLife
Volume12
Issue number1
DOIs
StatePublished - Jan 2022

Keywords

  • CRISPR-Cas9
  • Gene editing
  • HEK293T cells
  • Mitochondrial DNA
  • TFAM

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Biochemistry, Genetics and Molecular Biology(all)
  • Space and Planetary Science
  • Palaeontology

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