RESUMO

CRISPR-Cas9 is a method for genome editing that can be used efficiently for in vivo applications; the basic implementation of this method is used to generate genome site-directed sequence eliminations. Here we describe a protocol for genome editing using CRISPR-Cas9 in zebrafish (Danio rerio) one-cell embryos.

Assuntos

Sistemas CRISPR-Cas , Edição de Genes/métodos , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , DNA/isolamento & purificação , Embrião não Mamífero , Técnicas de Genotipagem/métodos , RNA Guia de Cinetoplastídeos , Peixe-Zebra/embriologia

RESUMO

Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish.

Assuntos

Fator de Ligação a CCCTC/genética , Desenvolvimento Embrionário/genética , Animais , Apoptose/genética , Sistemas CRISPR-Cas/genética , Sobrevivência Celular/genética , Cromatina/genética , Técnicas de Inativação de Genes/métodos , Humanos , RNA Mensageiro/genética , Peixe-Zebra