Dana Carroll's Lab Page
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We work on gene targeting. The basis of genetic analysis is the isolation and characterization of altered genomic sequences. Traditionally mutations have been isolated based on observed phenotypes, but with many whole genome sequences now available, investigators often want to generate designed mutations in specific genes of interest.
We have developed methods that allow the creation of highly specific localized mutations and targeted gene replacements. The basis for our approach is the use of zinc-finger nucleases (ZFNs). These hybrid proteins have a DNA-binding domain that can direct cleavage activity to a wide range of genomic sequences. Cleavage induces mutations via repair by nonhomologous end joining and gene replacement by homologous recombination with a manipulated donor DNA. ZFNs have been used successfully in many organisms and cell types from plants to humans.
In our lab we apply ZFNs to direct genome alterations in model organisms, particularly flies and worms. In Drosophila we have achieved very high frequencies of both mutagenesis and gene replacement — in the vicinity of 10% of all target alleles. Such efficiencies have allowed us to produce novel mutations in specific genes without reliance on a known phenotype. Our work with C. elegans is less advanced, but we have shown that ZFNs work well in this organism and promise to provide the tools for an effective gene targeting procedure. Since breaks in DNA stimulate both local mutagenesis and recombination in essentially all organisms, the ZFN approach is very broadly applicable.
Diagram of two ZFNs bound to a target sequence in the Drosophila yellow gene. Each zinc finger contacts three consecutive base pairs. The cleavage domains are green.
Example of a rosy mutation induced with ZFNs. The fly on the left has wild-type eye color; the one on the right is a rosy mutant.