Base Editing: A New Approach to Genome Editing

Abstract

Genome editing technologies, such as the recently discovered CRISPR/Cas9 system, allow the researcher to site-specifically modify genes of interest in living cells and organisms, and have the potential to yield new therapeutics to cure genetic diseases. Traditional Cas9-based strategies, however, rely on the generation of a double-stranded DNA break (DSB) and its subsequent repair. This method is inefficient at introducing precise nucleotide changes, and generates an excess of random insertions or deletions (indels). Considering that over half of known genetic diseases are caused by single nucleotide changes in DNA, new technologies that can cleanly correct such mutations are needed. This thesis outlines the development of base editing, a strategy for directly converting one base pair into another in living cells, without the generation of DSBs. Base editing uses a fusion protein composed of a cytidine deaminase, a catalytically impaired Cas9, and a base excision repair inhibitor to induce site-specific conversion of C to T or G to A in living cells. Base editing is able to correct a number of disease-relevant mutations in cell culture. New base editors with altered PAM specificities expand the number of disease mutations targetable in the human genome. Base editors were further engineered to improve their targeting precision and safety. The last part of the thesis aims to discover ways to improve the cytosolic delivery of macromolecules, a bottleneck for realizing their therapeutic potential. Previously, proteins fused to highly anionic moieties, such as (-30)GFP, have been delivered into cells using cationic lipids. To identify a more potent tag for protein delivery, we conducted a screen of highly anionic proteins from the human proteome for their ability to deliver cargo into cells with cationic lipids. We demonstrate that a relatively unknown protein called ProTα outperforms (-30)GFP in protein delivery assays.