KCS

We have developed novel methods for generating sequence-specific DNA-binding proteins that recognize predetermined DNA sequence elements. These DNA-binding proteins were then fused to domains that rendered the proteins capable of either up- or down-regulating the expression of genes whose promoter regions contain the target DNA sequences. To generate these so-called “designer” transcription factors, we first developed a modified one-hybrid selection method in yeast to isolate zinc finger domains with diverse DNA-binding specificities. We extensively screened zinc finger domains derived from sequences in the human genome and isolated 56 zinc fingers with distinct DNA-binding specificities. We used these zinc fingers as modular building blocks in the construct of novel, sequence-specific DNA-binding proteins. Fusion of these novel zinc finger proteins with either a transcriptional activation or repression domain yielded potent transcriptional activators or repressors, respectively. Our approach, termed GeneGrip technology, should facilitate the construction of customized DNA-binding proteins for use in research, medicine, and biotechnology.