"Design of Novel Homing Endonucleases as Site-Specific Genomic Cleavage Reagents "
Homing endonucleases (HEs) are a class of DNA cleaving enzymes that recognize long sequences of DNA with enough specificity to be used as site-specific cleavage reagents within a genome. The range of cleavable target sequences, however, is limited by the paucity of available HEs. This project aims to engineer novel HEs to cleave specific sequences within a cellular genome, and to investigate the phenotypic impact of this cleavage on the cell. The project uses existing crystallographic structural data of characterized HEs together with Rosetta, a protein structure prediction algorithm, to rationally design novel HEs to a DNA target of interest. The designed HEs are assembled and their cleavage activities are assayed in vitro and in vivo. Activity is refined by randomly mutating the designed HE and selecting or screening for especially active variants. Active variants are crystallized to understand the structural motifs that confer the new specificity.