Characterization of post-edited cells modified in the TFAM gene by CRISPR/Cas9 technology in the bovine model.

Vanessa Cristina De Oliveira,Clésio Gomes Mariano Junior,Marcos Roberto Chiaratti,José Ernesto Belizário,José Eduardo Krieger,Jean-Paul Concordet,Paulo Fantinato-Neto,Kelly Cristine Santos Roballo,Carlos Eduardo Ambrósio,Flávio Vieira Meirelles,Fabiana Fernandes Bressan

doi:10.1371/journal.pone.0235856

Abstract

Gene editing in large animal models for future applications in translational medicine and food production must be deeply investigated for an increase of knowledge. The mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters. This gene maintains mtDNA, and it is essential for the initiation of mtDNA transcription. Lately, we generated a new cell line through the disruption of the TFAM gene in bovine fibroblast cells by CRISPR/Cas 9 technology. We showed that the CRISPR/Cas9 design was efficient through the generation of heterozygous mutant clones. In this context, once this gene regulates the mtDNA replication specificity, the study aimed to determine if the post-edited cells are capable of in vitro maintenance and assess if they present changes in mtDNA copies and mitochondrial membrane potential after successive passages in culture. The post-edited cells were expanded in culture, and we performed a growth curve, doubling time, cell viability, mitochondrial DNA copy number, and mitochondrial membrane potential assays. The editing process did not make cell culture unfeasible, even though cell growth rate and viability were decreased compared to control since we observed the cells grow well when cultured in a medium supplemented with uridine and pyruvate. They also exhibited a classical fibroblastoid appearance. The RT-qPCR to determine the mtDNA copy number showed a decrease in the edited clones compared to the non-edited ones (control) in different cell passages. Cell staining with Mitotracker Green and red suggests a reduction in red fluorescence in the edited cells compared to the non-edited cells. Thus, through characterization, we demonstrated that the TFAM gene is critical to mitochondrial maintenance due to its interference in the stability of the mitochondrial DNA copy number in different cell passages and membrane potential confirming the decrease in mitochondrial activity in cells edited in heterozygosis.

Highlights

Mitochondria are intracellular organelles in charge of ATP synthesis, and they have their sole genetic material, the mitochondrial DNA [1, 2].The mtDNA is a circular double-stranded molecule essential for mitochondrial functioning and, for cellular performance
After 24 hours in culture, the cells started to adhere to plastic and presented fibroblastoid characteristics
The in vitro maintenance of the edited cells was achieved through the supplementation with uridine and pyruvate in the culture medium, the lack of supplementation resulted in cell death

Summary

Introduction

The mtDNA is a circular double-stranded molecule essential for mitochondrial functioning and, for cellular performance It encodes several subunits of the proteins in the electron transport chain and transporter and ribosomal RNAs [3]. It is replicated independently of nuclear DNA (nDNA), which codes the proteins responsible for mtDNA replication [4]. Among these various proteins, the mitochondrial transcription factor A (TFAM) is a protein that, after being encoded in the nucleus and translated it is exported to the mitochondria. It acts in the replication, transcription, and regulation of the number of copies of mtDNA [5,6,7]

Methods

Results

Conclusion