My research focuses on the optimization of targeted gene repair (TGR), a potential mechanism of gene therapy. In TGR, a DNA break is targeted near the site in the genome whose sequence is to be corrected. The break is then repaired by homologous recombination, using an exogenous DNA corresponding to the wild-type sequence. This is a low efficiency reaction in most cell types. My aim is to identify factors that enhance the efficiency of homologous recombination between the targeted genomic sequence and the exogenous DNA. Toward this end I have developed a GFP-based TGR reporter assay. I will be using this reporter to screen for increased TGR in cell lines over-expressing candidate recombination-promoting proteins. Additionally, we expect that the chromatin structure of the genomic locus will affect the efficiency of TGR. To address this issue I will be working with Jason Cummings to assess the effects of tethering chromatin-modifying enzymes to the site of TGR. Our colleagues in the Northwest Genome Engineering Consortium (NGEC) will use this information to optimize methods for effective gene therapy.
