Puesta a punto de la tecnología CRISPR para la edición génica de ADN mitocondrial en embriones de ratón

Alvarado Belinchón, Marta (2020). Puesta a punto de la tecnología CRISPR para la edición génica de ADN mitocondrial en embriones de ratón. Proyecto Fin de Carrera / Trabajo Fin de Grado, E.T.S. de Ingeniería Agronómica, Alimentaria y de Biosistemas (UPM), Madrid, España.

Description

Title: Puesta a punto de la tecnología CRISPR para la edición génica de ADN mitocondrial en embriones de ratón
Author/s:
  • Alvarado Belinchón, Marta
Contributor/s:
  • García Rebollar, Pilar
  • Bermejo Álvarez, Pablo
Item Type: Final Project
Degree: Grado en Biotecnología
Date: July 2020
Subjects:
Faculty: E.T.S. de Ingeniería Agronómica, Alimentaria y de Biosistemas (UPM)
Department: Producción Agraria
Creative Commons Licenses: Recognition - No derivative works - Non commercial

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Abstract

Mitochondria play fundamental metabolic and regulatory roles during mammalian preimplantation development, and mitochondrial deficiency can cause infertility in humans and livestock species. Oocyte’s mitochondrial DNA (mtDNA) amount has been positively correlated with embryonic developmental potential and a threshold under which embryo development is arrested has been proposed. Furthermore, mutations in mtDNA induce the development of mitochondrial diseases in 1/5000 births. These pathologies are often severe and multisystemic, especially affecting infants. In this work we have intended to eliminate or edit mtDNA in mice embryos by CRISPR/Cas 9 technology. mtDNA elimination could provide a model of mtDNA deficiency or mediate the elimination of mutated mtDNA copies, whereas edition could correct undesired mutations. In previous studies, it was observed that conventional CRISPR system, with nuclear targeting Cas 9, was unable to access mitochondrial DNA. Herein, we have generated a construct to synthesize mRNA encoding for Cas 9 fused with a Mitochondrial Targeting Signal (MTS) to be directed to mtDNA with two single-guided RNAs (sgRNAs). mRNA encoding for Cas 9: MTS and sgRNAs were injected into mice zygotes cytoplasm through microinjection. Zygotes injected with Cas 9: MTS alone were used as control for development. Both groups showed similar developmental rates (p > 0,05). In order to determine if the modified CRISPR system is able to reduce mtDNA quantity, we analysed it by quantitative PCR, observing no significant mtDNA reduction in the Cas 9: MTS + sgRNAs group compared to Cas 9: MTS alone (p > 0,05). Finally, we sequenced the target mitochondrial regions to determine if mtDNA edition had occurred, obtaining only wild-type sequences. These results suggest that the ribonucleoprotein composed by Cas 9: MTS and sgRNA is unable to access mtDNA, as no mtDNA elimination or edition occurred. Further research is needed to determine if the modified CRISPR system can be optimised for mtDNA edition.

More information

Item ID: 66709
DC Identifier: https://oa.upm.es/66709/
OAI Identifier: oai:oa.upm.es:66709
Deposited by: Biblioteca ETSI Agrónomos
Deposited on: 13 Apr 2021 10:09
Last Modified: 13 Jun 2021 22:30
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