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Dana Carroll

Professor and chair of biochemistry

B.A. Swarthmore College

Ph.D. University of California, Berkeley

Research

References

dana@biochem.utah.edu

Dana Carroll's Lab Page

Research

A powerful method for analysis of gene function is the isolation of mutations in the gene and evaluation of their effects on the form and function of the organism. Now with complete genome sequences available for many organisms, we can identify genes of interest with ease, and we would like to have methods to direct mutations specifically to a particular gene or gene segment. Even better, we want to make sequence changes of our choice, rather than ones that may occur naturally or with random mutagens. Using techniques of DNA manipulation, we can create altered sequences in the laboratory, and the challenge is to reintroduce them into their normal chromosomal locus by homologous recombination. This process is called gene targeting. It typically occurs only at low frequency, and it is possible in some organisms, but not many others.

We are building on the very general observation that double-strand breaks in DNA greatly stimulate recombination in their vicinity. We are using hybrid nucleases (ZFNs) comprised of a zinc-finger DNA-binding domain and a non-specific DNA-cleavage domain. Because zinc fingers can be manipulated to recognize a range of DNA sequences, the ZFNs can be directed to many different target genes. Because the cleavage domain must dimerize to cut DNA, we need to produce two different sets of zinc fingers to bind paired sites in close proximity (see Figure). An advantage of this requirement is that the cleavage reagent is assembled at the target, so random cleavage does not occur.

We are currently applying this technology to genuine chromosomal targets in a number of different organisms. In the fruit fly Drosophila melanogaster , we have demonstrated cleavage and mutagenesis by pairs of ZFNs at three different target genes: yellow, brown, and rosy . When a homologous donor sequence is provided, homologous recombination is greatly stimulated by ZFN cleavage, leading to targeted gene replacement in up to 15% of the progeny of treated flies. In collaboration with others, we have also achieved ZFN-induced cleavage and mutagenesis in the nematode Caenorhabditis elegans and the plant Arabidopsis thaliana . This approach should be applicable to a wide variety of targets in many different experimental organisms, where it promises to facilitate targeted mutagenesis. Ultimate applications to gene therapy in humans are easily envisioned.

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 left is a rosy mutant.

References