Scharenberg, Andrew MJacoby, Kyle Martin2013-11-142015-12-142013-11-142013Jacoby_washington_0250E_12139.pdfhttp://hdl.handle.net/1773/24223Thesis (Ph.D.)--University of Washington, 2013LAGLIDADG Homing Endonucleases (LHEs) are a family of highly specific DNA-cutting enzymes capable of recognizing target sequences of ~20 bp. In many eukaryotes, including humans and yeast, double-strand breaks induced by LHEs stimulate repair by Homologous Recombination, which can be used to alter or repair a gene if the template is supplied in trans, and Non-Homologous End Joining, which can be used to knock out a gene. The potential for such precise genome editing would reduce worry about insertional mutagenesis or misregulation, as only the specific gene under its native promoter would be targeted. Thus, LHEs have drawn intense interest for their research, biotech and clinical applications. Methods for rational engineering of LHEs have been limited by a small number of high quality starting enzymes, and an extremely restricted understanding of how to modify them to create novel enzymes that efficiently cleave hybrid target sequences. Here I describe my attempts to address these limitations by using a homology-directed search method to acquire, characterize, and engineer a robust set of I-OnuI-related LHEs which recognize a diverse set of target sequences. A system of iterative binding selection using yeast surface display was also developed to identify target sites for, and perform non-directed analysis of, previously uncharacterized enzymes. This diverse family of LHEs will serve both as a platform from which to launch short-distance designs, and a dataset to improve our understanding of protein-DNA interactions.application/pdfen-USCopyright is held by the individual authors.Gene therapy; Genome Engineering; Homing endonuclease; Meganuclease; Protein characterization; SELEXBiologyGeneticsmolecular and cellular biologyThesis