Development of Methods for Protein Delivery and the Directed Evolution of Recombinases

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Development of Methods for Protein Delivery and the Directed Evolution of Recombinases

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Title: Development of Methods for Protein Delivery and the Directed Evolution of Recombinases
Author: Thompson, David Brandon
Citation: Thompson, David Brandon. 2014. Development of Methods for Protein Delivery and the Directed Evolution of Recombinases. Doctoral dissertation, Harvard University.
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Abstract: As a class, protein-based therapeutics offer tremendous advantages over traditional small molecule drugs. Due to their sizes and folding energies, proteins are ideal for catalyzing chemical reactions, and can bind tightly and selectively to extended target surfaces. However, due to their large size, virtually all proteins are unable to spontaneously enter cells, and as a result protein therapeutics are restricted to extracellular targets. We developed a platform for delivery of proteins to intracellular target sites by engineering the surface chemistry of a model protein, green fluorescent protein (GFP). We found that 'supercharged' cationic GFP variants (scGFPs) bind to anionic cell surface molecules and initiate endocytosis, resulting in the efficient delivery of translationally fused cargo to intracellular targets. We discovered that scGFPs, and cationic delivery reagents in general, alter endosomal trafficking in a manner proportional to both their charge and their delivery efficiency, suggesting that avoidance of endosomal maturation is a key step in the endosomal escape of delivered protein cargos. We also developed a method for encapsulation of recombinant proteins by cationic lipid delivery reagents using negatively supercharged GFP.
Genetic modification technologies have matured rapidly following the discovery of protein classes with programmable DNA-binding specificities. While site-directed genetic knockout technologies are highly effective, targeted integration and repair remain comparatively inefficient. Site-specific recombinases directly catalyze strand exchange and ligation between DNA molecules, offering an approach to efficient genomic integration. However, most site-specific recombinases are not easily reprogrammable. To address this problem, we developed a genetic selection technique based on the Phage-Assisted Continuous Evolution (PACE) system, to enable the rapid evolution of recombinase proteins towards targets of interest. Using Cre recombinase as a model, the PACE system was optimized, validated, and used to evolve Cre variants with higher activity on their native loxP target site, as well as altered specificity towards a human genomic sequence within the hROSA26 locus.
Finally, we developed a method for enhancing the specificity of RNA-guided nucleases by restricting activity to sites of obligate dimeric nuclease assembly. We engineered a FokI nuclease fusion to a catalytically inactivated Cas9 protein that mediates efficient modification with significantly reduced off-target activity.
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