The Engineering and Evaluation of Genome Editing Tools in Mitochondria

  • Grace Mullally

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)


Mutations in mtDNA can have severe consequences for cellular energy
production and result in a severe class of incurable mitochondrial diseases. There is presently a lack of tools for the efficient editing of mitochondrial genomes which hinders study of mitochondrial biology and disease. Since 2013, CRISPR genome editing tools have had huge popularity and impact on molecular genetics due to their efficiency of targeting, versatility and ease of modification. Most studies using CRISPR have targeted the nuclear genome. This thesis details the first steps in the development of a suite of tools, which we have termed MitoCRISPR, broadening the current scope of CRISPR systems by tailoring them for the manipulation of mitochondrial genomes. Although we did not complete development of a MitoCRISPR system in this thesis, work detailed here sets out a framework for the project to move forward within.

Central to the development of a MitoCRISPR system is the targeted delivery of functional CRISPR machinery, namely a protein endonuclease and guide RNA, to the mitochondria. To this end, this thesis describes the cloning of a plasmid encoding mitochondrially targeted SpyCas9 and its expression in mammalian cells.

A library of modified CRISPR gRNAs were designed to incorporate RNA mitochondrial targeting aptamers. This thesis details findings that SpyCas9 gRNAs can be modified at several sites without impacting Cas9 complex formation and activity and investigates the import of gRNAs into whole cells and isolated mitochondria. Building on these findings, gRNA modifications which do impact Cas9 endonuclease activity and modified LbCas12a crRNAs
are also studied.

Given the development of tools for genome editing has real ethical implications, this thesis also details the development and findings from an art-science public engagement exercise through which we sought to open a dialogue with the Bristol-based public and gauge the perception of genome editing tools.
Date of Award23 Jan 2019
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorMark D Szczelkun (Supervisor) & Jon D Lane (Supervisor)

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