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

doi:10.3390/life12010022

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

Life (Basel). 2021 Dec 24;12(1):22. doi: 10.3390/life12010022.

Authors

Affiliations

¹ Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, São Paulo 13635-900, Brazil.
² Edward Via College of Osteopathic Medicine, Blacksburg, VA 24060, USA.
³ Department of Biomedical Science and Pathology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24060, USA.
⁴ Department of Genetics and Evolution, Federal University of São Carlos, São Carlos 13565-905, Brazil.
⁵ Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne 3052, Australia.
⁶ Department of Paediatrics, University of Melbourne, Melbourne 3010, Australia.
⁷ Stanley Manne Children's Research Institute, Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL 60611, USA.
⁸ Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, IL 1900, USA.
⁹ Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 1900, USA.
¹⁰ Laboratoire Structure et Instabilité des Génomes, Museum National d'Histoire Naturelle, INSERM U1154, CNRS UMR7196, 75231 Paris, France.

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.

Keywords: CRISPR-Cas9; HEK293T cells; TFAM; gene editing; mitochondrial DNA.

Abstract

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