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  • Book
    Jonathan Martin Geisinger.
    The ability to precisely modify the genome with high efficiency in a directed manner is dependent on generating and exploiting a double-strand break in DNA. The tools available to generate double-strand breaks include the recombinases and resolvases, the phage integrases, the homing endonucleases, zinc finger nucleases, transcription activator-like effector nucleases, and the CRISPR/Cas9 system, the most recently developed tool. These tools allow for in-depth studies of the function of genes and genome structure, the generation of organisms bearing economically important bio-synthetic pathways, and the development of novel gene and cell therapies for addressing previously untreatable diseases. Taking full advantage of each tool and knowing when to use each one requires a thorough understanding of how the tool functions. For most of the genome engineering toolkit, this understanding has been achieved. The CRISPR/Cas9 system, however, has been assumed to function in the same manner as the zinc finger and transcription activator-like effector nucleases despite generating blunt-end double-strand breaks rather than staggered breaks. This difference is important to understanding how Cas9-mediated double-strand breaks are repaired, and, thus, how best to exploit these breaks for genome engineering. This thesis illustrates the importance understanding how each genome engineering tool functions in developing advantageous methods for gene and cell therapies.