Knock-in of human TFAM gene in bovine fibroblasts cells by CRISPR/Cas9 technology

Abstract

The mitochondrial Transcription Factor A or TFAM is a member of the high mobility group proteins (HMGB) family that regulates mtDNA transcription, packaging, stability and replication. It binds to mtDNA promoters and plays an essential role in the mtDNA stress-mediated inflammatory response. Recently it was demonstrated that TFAM is required to maintain mtDNA integrity and that it is a key regulator of mtDNA copy number. The molecular mechanisms of mitochondrial inheritance are not yet fully elucidated, hence, in our study, we proposed to evaluate the possibility of editing the TFAM gene by CRISPR/Cas9 technology through the knock-in of the human TFAM gene in bovine fibroblasts seeking to understand mitochondrial inheritance and its possible ways of controlling it. For that, we will design the gRNA for the TFAM gene and optimize the knock-in protocol in bovine fibroblasts. The project developed during this internship will be continued in Brazil and may also generate new research projects. This study is crucial for understanding the segregation, inheritance and mitochondrial function as a model to elucidate the role of TFAM in the maintenance of mtDNA integrity and serve as a model for future gene therapies related to mitochondrial diseases.Key-words: TFAM, bovine, human, CRISPR/Cas9.