Abstract
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.
Highlights
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 mitochondrial DNA (mtDNA) promoters [1,2,3,4]
Our study proposed to edit the TFAM gene in bovine fibroblasts by CRISPR/Cas9 technology, firstly in order to assess if it’s possible to edit it by this tool, maintain the edited cells in culture since the embryonic TFAM disruption is lethal, and in the future characterize the generated cells and discover their application potential
Bovine fibroblasts used in this study were derived from a skin biopsy; the tissue was minced into small pieces and digested with collagenase type IV (Sigma C2674) for 3h at 37 ̊C
Summary
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 [1,2,3,4]. 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 essential for the initiation of transcription of mtDNA genes [5,6,7,8]. TFAM is a candidate gene for investigation of its functions in transcription and replication of mitochondrial DNA [1, 9]. TFAM is required to regulate the number of copies of mtDNA [10,11] and is essential for embryonic development in mice [12]. In bovine oocytes at different embryonic stages, the great importance of TFAM in the maintenance of the first stages of embryogenesis has been reported [13]
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