Genetic Engineering with Customizable Recombinases and Nucleases

The development of new tools that allow for the targeted modification of genomes is providing researchers with previously unattainable forms of control over diverse biological processes. Site-specific recombinases represent one such class of tools (Chapter 1). The rigid specificities of many site-specific recombination systems have limited their adoption in fields that require site-specific recombinase specificity to be easily customized, however. To address this problem, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities (Chapter 2). This stringent selection system is based on enzyme-mediated reassembly of the gene encoding β-lactamase. This approach enables identification of site-specific recombinase variants that broadly react with sequences within the human genome after only three rounds of selection. By combining this strategy with site-saturation mutagenesis, we show that site-specific recombinases with comprehensively re-designed target specificities can be created (Chapter 3). These enzymes recognize unnatural sequences >10,000-fold more effectively than their parental counterparts and catalyze targeted integration into the human genome with >80% specificity. We expand on these studies by combining substrate specificity analysis and directed evolution to develop a diverse collection of recombinase catalytic domains capable of recognizing an estimated 3.77 x 107 unique DNA sequences (Chapter 4). We show that zinc-finger recombinases (ZFRs) assembled from these re-engineered catalytic domains recombine user-defined DNA targets with high specificity and that designed ZFRs integrate DNA into targeted endogenous loci in human cells.;Site-specific nucleases are another class of enzymes that allow for the introduction of a broad range of custom alterations (Chapter 5). These customizable enzymes are based on programmable, sequence-specific DNA-binding modules linked to a non-specific DNA cleavage domain. In particular, zinc-finger nucleases (ZFNs) have enabled highly efficient gene disruption in numerous cell types and model organisms, and have facilitated the progress of targeted gene therapy in humans. However, contemporary DNA and mRNA-based ZFN delivery methods are associated with undesirable sideeffects that may limit the continued advancement of this technology. To address this problem, we investigated the direct delivery of purified ZFN proteins into cells (Chapter 6). We demonstrate the intrinsic cell-penetrating capabilities of ZFN proteins and show that direct delivery of ZFN proteins leads to highly efficient endogenous gene disruption in a variety of mammalian cell types. We also show that ZFN protein delivery leads to comparatively fewer off-target effects than ZFN-gene delivery systems that rely on expression from nucleic acids.;Together, these studies demonstrate the feasibility of generating customizable site-specific recombinases for targeted genetic engineering, and provide researchers with a new means for inducing targeted alterations in a safe and efficient manner.