Genome Engineering

For nearly a decade, the primary focus of our lab has been to develop robust, open-source reagents and methods that enable targeted genome engineering across many different cell types and model organisms. Primarily, this effort has involved the engineering and optimization of zinc finger nucleases (ZFNs), TALE nucleases (TALENs), and CRISPR/Cas9 nucleases. Using these three platforms, we (and our collaborators) have created designer genetic mutations with high efficiencies in zebrafish, plants, or human somatic and pluripotent stem cells. These mutations result from repair of nuclease-induced double-stranded DNA breaks by normal cellular repair processes (non-homologous end-joining or homologous recombination). Ongoing projects in the lab are aimed at defining and improving the specificity of these platforms and optimizing the editing capabilities of these methods for eventual therapeutic applications. We are also continuing to explore the development of novel technologies that will enable high-throughput genome editing.

 

Epigenome Engineering

Building off their success as nucleases, we have demonstrated that the utility of zinc fingers, TALEs, and CRISPR/Cas9 proteins can be extended so as to effect targeted changes in gene expression, a strategy we term "epigenome editing." In one strategy, we simply fuse the gene-activating VP64 or p65 domains to an engineered DNA binding domain targeted to an endogenous gene promoter to achieve gene activation. More recently, we have also shown that engineered TALEs can be used to direct histone modifications that can inactivate endogenous gene enhancers (work done with Bradley Bernstein's lab) and to direct demethylation of specific promoter CpGs that can lead to increases in endogenous gene expression. Notably, these studies provide important functional clues into the regulatory roles of epigenetic marks and cis-regulatory elements, and may enable more extensive studies functional analyses of such elements in the future. These capabilities will have important research applications for studying gene regulation and may also provide novel tools for altering the expression of disease-associated genes.