The long term goal of the proposed research is to engineer new, highly sequence-specific DMA bindingproteins with the ability to target and cleave specific human genes.
Our aim i s to generate and use newGene-Specific Reagents (GSR's) to promote the efficient recombinational repair or ablation of specific genesin living cells. Novel GSR's will be generated from preexisting homing endonuclease proteins of theLAGLIDADG family. These homing endonucleases catalyze the site-specific lateral transfer of parasitic DNAelements in all Kingdoms of life, and already possess many desirable properties for GSR engineering. For .example, LAGLIDADG homing endonucleases have long DNA target sites of 18-24 bp, a high degree ofDNA sequence specificity, and tight coupling of DNA site recognition to DNA strand cleavage. This lastproperty allows homing endonucleases to precisely target DNA strand cleavage to single phosphodiesterbonds in complex genomes with little or no 'collateral damage' as a result of off-target or spurious cleavageevents.In the proposed research we will generate LAGLIDADG homing endonuclease variants that target 9 humangenes and their canine or murine counterparts. These gene targets were chosen for their role in heritablehuman hematopoietic disease; in heritable immune deficiency syndromes; or in tyrosinemia type I, one of themost common heritable inborn errors of metabolism. Our experimental Aims are:
Aim 1 : Generate optimized LAGLIDADG homing endonuclease proteins for mammalian genome engineeringAim 2: Develop gene-specific targeting variants of LAGLIDADG homing endonucleases directed againsthuman disease gene targetsAim 3: Determine ability of engineered, gene-specific LAGLIDADG variant proteins to catalyzerecombinational gene repair or gene inactivation in vivo.
