Grant: Discovery Grants Program - Individual

My program of research deals with mobile genetic elements, discrete units of DNA that promote their own spread within and between genomes. For the past ten years, my laboratory has studied the evolution and function of homing endonucleases, site-specific DNA endonucleases that spread by making a double-strand break (or nick) in a DNA target. Over the last funding period, we focused on basic mechanistic questions concerning DNA cleavage by the GIY-YIG family of homing endonucleases, and translating this mechanistic understanding to the generation of novel monomeric chimeric nucleases for genome-editing applications. Academic and industrial researchers use genome-editing reagents to introduce precise modifications in complex genomes for applications in agriculture, aquaculture, synthetic biology, and discovery-based research in model organisms. Our long-term goal is to develop a set of monomeric nucleases with tailored cleavage specificities for genome editing and synthetic biology applications that require high fidelity. Monomeric domains are useful for genome-engineering applications because a monomeric nuclease domain requires that only a single chimeric nuclease need be designed to target a given sequence. Moreover, many monomeric nuclease domains have inherent cleavage specificity that will limit off-target cleavage and cellular toxicity. Understanding the molecular basis of cleavage specificity will enable more predictable targeting of the domains to desired sequences, as well as identifying potential off-target sites. This will be achieved by a combination of genetic selections and protein-DNA crosslinking studies to identify residues that contribute to DNA cleavage specificity. For the GIY-YIG enzymes, a long term goal will be structural studies of GIY-YIG homing endonucleases bound to a DNA substrate, as this will give us insight into residues that contribute to cleavage specificity. Towards this goal, we have initiated a collaboration with Dr. Murray Junop (Western University). In summary, my research program addresses fundamental questions concerning structure and function of site-specific DNA endonucleases, in addition to biotechnological applications that derive from these studies. Given the widespread use of genome-editing nucleases in academic and industrial settings, our research program is well positioned to make important contributions in the development of critical reagents.