Date Published: March 7, 2019
Publisher: Public Library of Science
Author(s): Vanessa Cristina de Oliveira, Gabriel Sassarão Alves Moreira, Fabiana Fernandes Bressan, Clésio Gomes Mariano Junior, Kelly Cristine Santos Roballo, Marine Charpentier, Jean-Paul Concordet, Flávio Vieira Meirelles, Carlos Eduardo Ambrósio, Zhi-Yao He.
The mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA) binding protein essential for the initiation of transcription and genome maintenance. Recently it was demonstrated that the primary role of TFAM is to maintain the integrity of mtDNA and that it is a key regulator of mtDNA copy number. It was also shown that TFAM plays a central role in the mtDNA stress-mediated inflammatory response. In our study, we proposed to evaluate the possibility of editing the TFAM gene by CRISPR/Cas9 technology in bovine fibroblasts, as TFAM regulates the replication specificity of mtDNA. We further attempted to maintain these cells in culture post edition in a medium supplemented with uridine and pyruvate to mimic Rho zero cells that are capable of surviving without mtDNA, because it is known that the TFAM gene is lethal in knockout mice and chicken. Moreover, we evaluated the effects of TFAM modification on mtDNA copy number. The CRISPR gRNA was designed to target exon 1 of the bovine TFAM gene and subsequently cloned. Fibroblasts were transfected with Cas9 and control plasmids. After 24 h of transfection, cells were analyzed by flow cytometry to evaluate the efficiency of transfection. The site directed-mutation frequency was assessed by T7 endonuclease assay, and cell clones were analyzed for mtDNA copy number by Sanger DNA sequencing. We achieved transfection efficiency of 51.3%. We selected 23 successfully transformed clones for further analysis, and seven of these exhibited directed mutations at the CRISPR/Cas9 targeted site. Moreover, we also found a decrease in mtDNA copy number in the gene edited clones compared to that in the controls. These TFAM gene mutant cells were viable in culture when supplemented with uridine and pyruvate. We conclude that this CRISPR/Cas9 design was efficient, resulting in seven heterozygous mutant clones and opening up the possibility to use these mutant cell lines as a model system to elucidate the role of TFAM in the maintenance of mtDNA integrity.
The mitochondrial transcription factor A (TFAM) is a member of the High Mobility Group Box (HMGB) subfamily structurally composed of 2 HMGB domains, HMG1 and HMG2, which binds to mtDNA promoters [1–4]. The TFAM gene plays an important role in cellular physiology involved in the maintenance of mtDNA, and regulates the number of mtDNA copies. It is also essential for the initiation of transcription of mtDNA genes [5–8].
Our study protocol was approved by the Research Ethics Committee (Approval No. 5828250215) of the Faculty of Animal Science and Food Engineering, University of São Paulo, Brazil.
Genetic editing in cattle is an important tool for generating gene knockouts in animal models, such as in herds for pharmaceutical purposes. These genetic modifications are of extreme importance for both agricultural science and biomedical applications, rendering this particular animal model more suitable for gene therapy when compared to laboratory rodent models [5,17–19].