Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

David Edgell

Abstract

Manipulation of complex genomes has many beneficial downstream applications in agriculture and human gene therapy. Precise genome-editing requires the introduction of a specific DNA double-stand break at a locus of interest, in turn inducing host DNA repair pathways to cause gene knockout through non-homologous end-joining or gene repair using homologous recombination and donor template. No matter the application, the field has depended on a few reagents to introduce precise double-strand breaks in host genomes. LAGLIDADG homing endonucleases or meganucleases harness the natural properties of these rare-cutting enzymes to target precise sequences in a complex genome. Other successful reagents are derived from a type IIS restriction endonuclease, FokI, fused to various DNA-binding architectures such as zinc finger domains and transcription activator-like effector domains. However, the discovery of clustered regularly interspaced short palindromic repeat-associated protein, CRISPR-Cas9, has dominated the field with its ease of design requiring a single RNA molecule to target the sequence of interest. Even with a handful of reagents to choose from, no one reagent is suitable for every application as every reagent has its own set of limitations and advantages.

Here we present another potential genome-editing reagent derived from a GIY-YIG homing endonuclease, I-TevI, fused to all four DNA-targeting proteins described above. First, we demonstrate that I-TevI is a portable nuclease domain that can be targeted using Zinc-Fingers and LAGLIDADG proteins. Using these new reagents, we were able to further characterize I-TevI specificity using high throughput in vitro and in vivo screens to highlight important sequence requirements for targeting. Using this knowledge, we systematically engineered new I-TevI variants with altered specificity to broaden the number of targets available for I-TevI-derived reagents. We incorporated these new I-TevI variants into a more versatile dual-active nuclease, TevCas9, capable of introducing two double-strand breaks at a single target site. This dual cleavage event is capable of excising a short DNA fragment from the target site and is unique to I-TevI derived fusions. We envisioned that the monomeric, sequence-specific I-TevI catalytic domain would improve current tools by providing additional specificity and the ability to introduce dual-cleavage event would present unique applications for genome engineering.

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