Improving Zinc Finger Nucleases - Strategies for Increasing Gene Editing Activities and Evaluating Off-Target Effects

Abstract

Zinc finger nucleases (ZFNs) induce double-strand DNA breaks at specific recognition sites. ZFNs can dramatically increase the efficiency of incorporating desired insertions, deletions, or substitutions in living cells. These tools have revolutionized the field of genome engineering in several model organisms and cell types including zebrafish, rats, and human pluripotent stem cells. There have been numerous advances in ZFN engineering and characterization strategies, some of which are detailed in this work. The central theme of this dissertation is improving the activity and specificity of engineered zinc finger nucleases with the ultimate goal of increasing the safety and efficacy of these tools for human therapy. As a first step, I undertook a large-scale effort to demonstrate that the modular assembly method of ZFN synthesis has a significantly higher failure rate than previously reported in the literature. This strongly suggested that engineering of ZFNs should better account for context-dependent effects among zinc fingers. The second advance reported in this dissertation is a method for biasing repair of zinc finger protein-induced DNA breaks toward homology-driven rather than error-prone repair in the presence of a donor template. Catalytically inactivating one monomer of a ZFN dimer results in a zinc finger nickase (ZFNickase) whose cleavage preference is directed at only one DNA strand. In human cell reporter assays, these ZFNickases exhibit a higher likelihood of repair by homology-driven processes, albeit with reduced absolute rates of correction. With further optimization, zinc finger nickases could provide a safer alternative to ZFNs in the context of gene correction therapies. Third, realizing there was no robust method for determining off-target cleavage sites of ZFNs in a genome-wide manner, I validated a collaborator’s novel in vitro selection system in human cells by identifying eight new potential off-target cleavage sites for a ZFN pair currently being used in clinical trials. Although it is unlikely these low-frequency mutations would be deleterious to patients, these results demonstrated that ZFNs induced more off-target effects than had been appreciated by previous work in the field. Collectively, the findings of this dissertation have contributed to more robust strategies for designing and evaluating ZFNs.