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Download (8MB) Kentner, Jeffrey Louis (2015) Engineering the zinc finger recombinase for use in targeted genomic editing. PhD thesis. https://theses.gla.ac.uk/6910/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] Engineering the Zinc Finger Recombinase for use in Targeted Genomic Editing Jeffrey Louis Kentner Honours Bachelor of Science Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Institute of Molecular, Cell and Systems Biology College of Medical, Veterinary and Life Sciences University of Glasgow November 2015 Abstract The zinc finger recombinase (ZFR) is a chimeric enzyme system for use in targeted genomic editing. The ZFR is comprised of a recombinase catalytic domain, which is able to catalyse recombination reactions between DNA molecules, and a zinc finger array DNA- binding domain, which is able to target the enzyme to a desired genetic sequence. Currently the ZFR is in an early stage of development and will require several crucial improvements before it can be adopted as a useful genome editing tool by researchers. Two major challenges involve two important parameters of ZFR-catalysed integration reactions: specificity of the orientation of the integration, and stability of the integration. Currently, the ZFR system is unable to select the orientation of integrations, and because the recombination reactions are reversible, the integrations are not stable (and, in fact, are stochastically disfavoured). This project aimed to impart the ZFR system with the ability to perform stable, orientation-specific integrations. In order to achieve the project aims, the experiments of this project sought to generate new pairs of ZFR mutants that were differentially modified at either their catalytic domain or DNA-binding domain, and characterize their behaviour in an E. coli- based recombination assay. The overall strategy was to exploit the interactions within the protein-protein interfaces of the ZFR tetramer to produce selective compatibility, and to generate differences in enzyme activity when two mutants (one active, and one inactive or less active) were either paired as heterodimers or as homodimers. During ZFR recombination reactions heterodimers rearrange to form homodimers, and thus the production of a significant difference in activity between heterodimers and homodimers represents a recombination reaction directionality bias. Both the catalytic domain modification and DNA-binding domain modification approaches proved able to produce the desired bias in the directionality of ZFR recombination reactions, which is predicted to lead to both stability of an integration, and specificity of integration orientation through a stochastic process. Of particular note, was a strategy utilizing a heterodimer that consisted of one ZFR subunit targeted specifically to the DNA, paired with a recombinase subunit (with its native DNA-binding domain) targeted non-specifically to the DNA. The difference in activity between these subunits paired in heterodimer and homodimer configurations appeared to produce a completely irreversible recombination reaction 2 without any apparent reduction in recombination reaction efficiency. Furthermore, the results of the catalytic domain modification and DNA-binding domain modification experiments suggest that it should be possible to generate a combination strategy in which the recombinase subunit with the native DNA-binding domain is catalytically inactive unless operating within an intended heterodimer, overcoming the potential problem of unwanted off-target activity from homodimers of this subunit. The success of this work in producing ZFR reactions with the potential to catalyse stable, orientation-specific integration reactions potentially represents a major leap forward in ZFR research; however, these results must be further validated in a mammalian cell system. Although genome editing systems such as the CRISPR-Cas9 RNA- guided endonuclease now allow researchers to modify genomes within embryos and cell culture with ease, off-target effects, reliance on endogenous homology directed repair (HDR) activity, unfavourable ratios of HDR to non-homologous end joining (NHEJ) activity at the target site, and low efficiency make targeted endonuclease technology impractical for use in in vivo gene therapy applications and genome editing in some cell types (e.g. non-dividing cells such as neurons and myocytes). Therefore, the ZFR is envisioned as a genome editing tool that can fill this vacant niche for gene editing in non-dividing cell types and human in vivo gene therapy. 3 Table of contents Abstract .................................................................................................................................. 2 Table of contents ................................................................................................................... 4 List of tables .........................................................................................................................12 List of figures ........................................................................................................................13 Accompanying material .......................................................................................................16 Acknowledgement ...............................................................................................................17 Author's declaration .............................................................................................................18 Abbreviations .......................................................................................................................19 Chapter 1: Introduction........................................................................................................22 1.1 The dream of targeted genomic sequence editing ...............................................22 1.2 Current gene therapy ............................................................................................22 1.2.1 Gene therapy background .............................................................................22 1.2.2 Gene therapy characteristics .........................................................................23 1.2.3 Targeted genome editing enzymes for gene therapy ....................................24 1.3 A brief history of targeted genomic sequence editing ..........................................25 1.3.1 The development of gene targeting ..............................................................25 1.3.2 'Classical' targeted gene repair ......................................................................31 1.3.3 Adeno-associated viruses (AAVs) ...................................................................33 1.4 Where we're at today: current genome editing techniques.................................34 1.4.1 Programmable site-specific nucleases and nickases .....................................34 1.4.2 Engineered homing endonucleases ...............................................................38 1.4.3 Chimeric restriction enzymes with programmable binding domains: ZFNs and TALENs ...................................................................................................................41 1.4.4 RGENs: the CRISPR-Cas9 system ....................................................................45 1.4.5 Delivery of site-specific nucleases .................................................................49 4 1.4.6 Limitations of the site-specific nuclease approach ........................................51 1.5 Chimeric recombinases with programmable binding domains: ZFRs and TALERs 54 1.5.1 Introduction ...................................................................................................54 1.5.2 ZFR studies to date .........................................................................................59 1.5.3 ZFR system parameters and outcomes: dimer-dimer orientation specificity and reaction directionality ...........................................................................................65 1.6 Tn3 resolvase .........................................................................................................68 1.6.1 Origin ..............................................................................................................68 1.6.2 Structure of Tn3 resolvase .............................................................................68 1.6.3 Crystal structures of γδ resolvase ..................................................................69 1.6.4 Hyperactive Tn3 resolvase mutants ...............................................................71 1.6.5 'Primary', 'secondary', and 'tertiary' activating mutations ............................72 1.7 Binding domains ....................................................................................................74 1.7.1 Zinc finger arrays ............................................................................................74
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