Generación de un modelo de deficiencia mitocondrial mediante la tecnología CRISPR

López Palacios, Alba (2019). Generación de un modelo de deficiencia mitocondrial mediante la tecnología CRISPR. Proyecto Fin de Carrera / Trabajo Fin de Grado, E.T.S. de Ingeniería Agronómica, Alimentaria y de Biosistemas (UPM), Madrid.

Description

Title: Generación de un modelo de deficiencia mitocondrial mediante la tecnología CRISPR
Author/s:
  • López Palacios, Alba
Contributor/s:
  • Bermejo Álvarez, Pablo
  • Pascual Bañuls, Laura
Item Type: Final Project
Degree: Grado en Biotecnología
Date: June 2019
Subjects:
Faculty: E.T.S. de Ingeniería Agronómica, Alimentaria y de Biosistemas (UPM)
Department: Biotecnología - Biología Vegetal
Creative Commons Licenses: Recognition - No derivative works - Non commercial

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Abstract

Mitochondria are cellular organelles that serve essential functions such as the obtention of energy in form of ATP (adenosine triphosphate) through oxidative phosphorylation. Mitochondria are the most abundant organelles in oocytes. During preimplantation development, they play crucial roles on embryo metabolism, calcium homeostasis and apoptosis signalling, among others. Mitochondria contain their own genome which can be mutated hampering its functionality. As each oocyte contains multiple mitochondria, functional and mutated mitochondria may coexist leading to a phenomenon termed heteroplasmy. Heteroplasmy may have severe consequences for embryo or fetal development. These consequences depend on the percentage of mutated mitochondria and the severity of the phenotype generated by each specific mutation. Genome edition opens a way to correct heteroplasmy, as it may be directed to eliminate those mitochondria harbouring deletereous alleles or to correct the mutation. However, currently it is unclear whether conventional CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9) system, where Cas9 contains a nuclear localization signal is able to recognize a DNA target located on the mitochondrial genome. The objective of this study has been to test whether conventional CRISPR/Cas9 system can edit mitochondrial DNA. For that aim we have targeted two different mitochondrial DNA sequences, allocated on the genes CYTB (cytochrome b) and ND4 (NADHubiquinone oxidoreductase chain 4) by ooplasm microinjection of CRISPR/Cas9 components in murine zygotes. Following microinjection, control embryos (microinjected with Cas9-encoding mRNA) or embryos injected with Cas9 mRNA and sgRNA against CYTB or ND4 genes were cultured in vitro to the blastocyst stage. Developmental rates were similar between the three groups. Genome edition analysis shown that the target sequences remained unaltered. Finally, to precisely determine whether CRISPR had induced a depletion in mitochondrial DNA, we quantified mitochondrial DNA in these three groups by quantitative PCR, obtaining no differences between groups on this parameter. These results show that conventional CRISPR/Cas9 system is not able to reach and recognize mitochondrial genome, as no mitochondrial DNA edition or depletion occurred.

More information

Item ID: 57525
DC Identifier: http://oa.upm.es/57525/
OAI Identifier: oai:oa.upm.es:57525
Deposited by: Biblioteca ETSI Agrónomos
Deposited on: 16 Dec 2019 13:42
Last Modified: 16 Dec 2019 13:42
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