Use of RecA to Promote Gene Targeting

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DESCRIPTION (provided by applicant): The forward genetic screens conducted in zebrafish have firmly established this system as a model for human disease and development. Sequencing of the zebrafish genome and the development of powerful genomics-based tools have also contributed to the placement of this model system at the forefront of vertebrate developmental genetics. Despite this prominent role for zebrafish, the ability to fully utilize genomics- based tools in this model system is limited by the inability to perform reverse genetics. To address this need, we propose to develop simple methods that most labs with moderate molecular biology skills could use to disrupt or target genes. Our method uses a significantly modified version of the bacterial recombinase protein RecA that has greatly enhanced activities. RecA is a multifunctional enzyme involved in recombination and the repair of double-strand breaks. RecA has diverse enzymatic functions, including the ability to coat single-stranded DNA, to carry out homology searches, and to promote strand exchange during recombination. We propose to use our modified version of RecA to coat single stranded DNA and produce DNA-RecA filaments that are homologous to the gene we wish to target. In Aim 1, we will test whether these filaments can create small deletions and insertions in the zebrafish genome. In Aim 2, we will determine the frequency with which the single-stranded DNA-RecA filaments promote targeted insertion of a reporter gene into the zebrafish genome. Both of the activities described in the above two aims would be consistent with the ability of the single- stranded DNA-RecA filaments to create double strand breaks. We provide significant preliminary results to support both of these aims and demonstrate how injection of chimeric versions of RecA proteins can mediate gene targeting in mitotic cells of the early zebrafish embryo and in the zebrafish germline. While these techniques are being developed in zebrafish, we believe they can be adapted for genome modifications in most models for human disease and will have applications for human gene therapy. PUBLIC HEALTH RELEVANCE: We propose to develop methods in zebrafish that utilize the bacterial recombinase protein RecA to create site directed modification of the genome. These techniques have the potential to revolutionize reverse genetic approaches in all animal models of human disease. Successful completion of this proposal will have a profound impact on the identification of pharmaceutical targets for drug discovery and the production of new animal models of human disease.