Zebrafish genome editing through CRISPR / Cas9 for generation of translational model of tissue regeneration

Abstract

The limited regenerative capacity of mammals has serious implications for tissue damage. Throughout the life cycle, loss of cells in long-lasting tissues such as brain, heart, skeletal muscles and thymus occurs due to the aging process itself and complications due to tissue damages such as amputation and cardiovascular diseases that can not be repaired by the patients tissue regeneration. In the period 2008-2018, 528,703 amputation procedures related to lower and upper limbs were registered in Brazil, according to the SIHSUS database. The attempt to recapitulate the regenerative capacity of urodell amphibians in mammals has employed approaches such as stem cells, gene therapy and tissue engineering. However, the most recent advances are still limited to the phalanges of the mouse digits. Animal models have been used in translational research to study possible solutions for tissue regeneration studies. It is known that evolutionarily conserved genes are involved in limb or limb regeneration. This suggests an innate ability to regenerate tissues lost in most animals and that differences in regenerative abilities are not related to the presence or absence of regeneration genes but to the regulatory mechanisms that control the activation of these genes after injury. Genes of the Wnt / ²-catenin and BMP pathways and the transcription factors Runx2 and Osterix control the regeneration process. Sequencing and epigenetic techniques combined with traditional transgenic assays have uncovered numerous genetic enhancers associated with regeneration. Using modern genomic editing techniques based on CRISPR / Cas9 and the high regenerative potential to reconstruct damaged tissues and structures such as fins, heart and nerve tissue, zebrafish is an important translational model in tissue regeneration research. This study aims to introduce specific lesion enhancers as LEN through the CRISPR / Cas9 technique in the upstream regions of the regeneration control genes in zebrafish. Because they do not induce expression during embryonic development and during other stages of development, these enhancers diminish the potential negative effects of overexpression of the genes of interest. Given the versatility of the zebrafish model and its use as an animal model for translational research, our objective is to investigate the overexpression of these injury-induced regeneration genes to investigate potential improvements in regenerative capacity using CRISPR / Cas9 technology. In this way this proposal can find a possible solution of gene therapy for the tissue regeneration problem in humans. In this sense, the states of RS and SP are poles of knowledge production in the area of Biotechnology, having several researchers and a range of graduate programs at the level of excellence. This proposal proposes a biotechnological solution to a health problem. We hope that at the end of this project, scientific articles and patents of invention will be produced that can be transferred to health companies or Startups that can develop the full technology and bring it to the market by generating royalties for the RS and SP. In addition, the researchers are highly engaged in the Innovation activities of the states of SP and RS. UFPel and UNESP have already carried out technology transfers and have experience in the area of agreements and contracts with industry. Also, the present proposal has experienced researchers in the area of transgenesis in zebrafish of Brazil and Spain in the team, characterizing the internationalization of the proposal. The laboratories are equipped to carry out the project. Also, no previous national and international patent banks were found regarding the specific theme of the proposal, demonstrating its technological viability. (AU)