kjacoby at uw dot edu
High throughput discovery and detailed characterization of homing endonucleases for targeted gene modifications
Highly specific, rare cleaving nucleases are currently being investigated for their use in targeted gene repair and disruption. Among these, homing endonucleases show great promise due to their compact size, monomeric nature, and coupled recognition and catalytic activities. Unfortunately, they are not as modular as competing technologies such as TAL Effector- or Zinc Finger-coupled nucleases. Since homing endonucleases with multiple mutations (aimed at redirecting their target sequence) tend to produce ineffective enzymes, it is of utmost importance that we explore target sites recognized by novel homologs. My work to massively expand the accessible sequence diversity will reduce the number of required mutations needed to access a given target sequence goal.
In order to validate binding and cleavage activities of a putative homing endonucleases, I must first know their target sequence. Unfortunately, available sequence data from which the homologs were derived do not always contain engough information to determine their corresponding recognition sequence. I have therefore integrated a first principals target determination protocol (SELEX) into our nuclease validation and characterization platform (yeast surface display). This marriage of technologies not only aims to identify previously unknown target sites, but also expands our high throughput testing capabilities, providing higher resolution binding and cleavage profiles than have ever been able to be obtained.
By the completion of my thesis, I hope to provide the scientific community with a large array of carefully characterized homing endonucleases capable of accessing any given locus via minimal modifications. The resulting enzymes could therefore be quickly generated and would be able to function in vivo robustly with minimal off-target effects. These technologies aim to open the doors to performing complex genetic manipulations on organisms with large genomes for clinical, biotechnological, and research purposes; gene therapy in humans, for example.
Also, see my molecular medicine certificate program profile.
Copyright © 2003-2013 Molecular & Cellular Biology Program, University of Washington
Fred Hutchison Cancer Research Center | University of Washington
Institute for Systems Biology | Seattle Biomed