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Edition of TFAM gene by CRISPR/Cas9 technology in bovine model

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Autor(es):
de Oliveira, Vanessa Cristina [1] ; Alves Moreira, Gabriel Sassarao [1] ; Bressan, Fabiana Fernandes [1] ; Mariano Junior, Clesio Gomes [1] ; Santos Roballo, Kelly Cristine [1] ; Charpentier, Marine [2] ; Concordet, Jean-Paul [2] ; Meirelles, Flavio Vieira [1] ; Ambrosio, Carlos Eduardo [1]
Número total de Autores: 9
Afiliação do(s) autor(es):
[1] Univ Sao Paulo, Fac Anim Sci & Food Engn, Dept Vet Med, Sao Paulo - Brazil
[2] Museum Natl Hist Nat, CNRS UMR7196, Lab Struct & Instabilite Genomes, INSERM U1154, Paris - France
Número total de Afiliações: 2
Tipo de documento: Artigo Científico
Fonte: PLoS One; v. 14, n. 3 MAR 7 2019.
Citações Web of Science: 0
Resumo

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. (AU)

Processo FAPESP: 17/08896-4 - Potencial de utilização de células pós-editadas no gene TFAM pela tecnologia CRISPR CAS 9 em modelo bovino
Beneficiário:Vanessa Cristina Oliveira Nogueira de Pontes
Linha de fomento: Bolsas no Brasil - Pós-Doutorado
Processo FAPESP: 13/08135-2 - CTC - Centro de Terapia Celular
Beneficiário:Dimas Tadeu Covas
Linha de fomento: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs