Zinc finger nucleases (ZFNs) are customizable nucleases that have been used to alter endogenous genes in Drosophila, plant, and human cells with high efficiencies ranging from 1%-50%. ZFN-induced DNA double- stranded breaks (DSBs) can be repaired by error-prone non-homologous end-joining (NHEJ) or by homologous recombination (HR) with an appropriately designed exogenous """"""""donor template"""""""" to introduce specific mutations near the break. Recent proof-of-principle experiments have shown that ZFNs can also be used to create targeted NHEJ-mediated knockout mutations in zebrafish embryos. These new findings raise important questions about how to make engineered ZFN technology widely available to zebrafish researchers and whether ZFNs can be used to induce highly efficient HR-mediated gene targeting in zebrafish. Long-term goals of the proposed research are to improve the quality and scope of genetic tools available for use in zebrafish and to make customized ZFN technology readily accessible to all zebrafish research laboratories. To accomplish these goals, we are combining the zinc finger engineering and zebrafish expertise of the Joung and Peterson labs, respectively, at Massachusetts General Hospital to propose the following Specific Aims: (1) to engineer and to make publicly available ZFN pairs for 500 different zebrafish genes using a rapid, robust, and highly effective zinc finger engineering method known as OPEN (for Oligomerized Pool Engineering);(2) to develop and validate a faster and simplified version of the OPEN method (OPEN version 2.0) so that it can be used by any interested academic laboratory;and (3) to demonstrate that ZFNs can be used to induce highly efficient HR-based gene targeting in zebrafish and to optimize parameters for this application. Successful completion of these aims will broaden the use and application of ZFNs for genetic studies in zebrafish, an established and important model organism for studying human development and disease.
Zinc finger nucleases (ZFNs) are customizable """"""""molecular scissors"""""""" that can be used to alter specific genes in a variety of organisms including zebrafish. This proposal will seek to enable broad adoption of ZFN technology by the academic zebrafish community and to expand the range of genome modifications that can be performed with ZFNs. Successful completion of the aims of this proposal will improve the quality and scope of genetic tools available for use in zebrafish, an important model organism for studying human development and disease.
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