S151: Novel tools for genome engineering

Genome engineering focuses on cellular engineering at the genomic level and is a rapidly growing area of academic and industrial importance.  Here I report our recent work in the development of novel tools for engineering mammalian genomes for therapeutic applications.  The first set of tools is ligand-regulated gene expression systems that can significantly broaden the clinical efficacy and safety as well as the application range of gene therapy.  We used a combined directed evolution and rational design approach to engineer variants of human estrogen receptor alpha ligand binding domain that bind and respond to two synthetic ligands with high specificity and potency.  The resulting orthogonal receptor-ligand pairs exhibited nanomolar or subnanomolar response to their respectively matched synthetic ligand, while showing negligible or no activation in response to the natural ligand.  The second set of tools is engineered artificial nucleases with novel defined sequence specificity for gene targeting.  We developed a new class of artificial DNA nucleases, so called TAL effector nucleases (TALENs), in which the central repeat DNA binding domain of the TAL effectors is fused with the non-specific DNA cleavage domain of the FokI restriction endonuclease through a flexible linker. As proof of concept, we constructed a pair of custom-designed TALENs to target a sequence within human β-globin (HBB) gene locus. This pair of TALEN with the optimized scaffolds allowed for efficient cleavage at the desired site in human cells and enhanced the gene targeting rate by >1000-fold with no detectable cytotoxicity.