J. Keith Joung

Our laboratory has developed robust protein engineering methods for constructing artificial “designer” DNA-binding domains. Our efforts focus on Cys2His2 zinc fingers, the most common structural motif found in eukaryotic transcription factors.  We are also developing engineered zinc finger proteins for applications in biological research and gene therapy.

Engineering “designer” zinc fingers with novel DNA-binding specificities
The Joung lab is a founding member of The Zinc Finger Consortium (http://www.zincfingers.org). As part of this collaborative effort, we have developed a robust, “open-source,” selection-based method for rapidly engineering synthetic zinc finger domains with novel, defined DNA-binding specificities (Maeder et al., 2008; Maeder et al., 2009).  Designer zinc fingers made using this approach (termed OPEN for Oligomerized Pool ENgineering) can be used to target functional domains to specific genomic loci in cells and have numerous applications in a broad range of cell types (see below).  In addition, analysis of the amino acid sequences and specificities of these artificial fingers will be useful for developing algorithms to predict the DNA-binding specificities of naturally occurring zinc finger domains.

Engineered zinc finger nucleases for targeted, highly efficient genome manipulation
Zinc finger nucleases (ZFNs), consisting of designer zinc fingers fused to a non-specific endonuclease domain, can be used to introduce targeted DNA alterations with high efficiency at specific genomic loci.  These alterations result from repair of ZFN-induced double-stranded DNA breaks by normal cellular repair processes (non-homologous end-joining or homologous recombination).  ZFNs made using our OPEN method can be used to create targeted endogenous gene mutations with high efficiency in zebrafish (Foley & Yeh et al., 2009), plants (Townsend et al., 2009), and human somatic, ES, and iPS cells (Zou et al., 2009; Maeder et al., 2008).  On-going projects in the lab are aimed at further optimizing the ZFN genome modification methodology and using it for applications in biological research and for gene therapy of monogenic diseases. 

Altering cellular phenotypes using combinatorial zinc finger transcription factor libraries
We have recently constructed large combinatorial libraries of zinc finger domains which can be fused to various transcriptional regulatory domains (e.g.--activation or repression domains).  We are introducing these libraries into human cells to induce specific desired phenotypes and cellular states.