ADVANCING ZINC FINGER NUCLEASES FOR TARGETED GENOME MANIPULATION

描述（由申请人提供）：锌指核酸酶（ZFNs）是一种可定制的核酸酶，用于改变果蝇、植物和人类细胞中的内源性基因，效率在1%-50%之间。zfn诱导的DNA双链断裂（DSBs）可以通过易出错的非同源末端连接（NHEJ）或同源重组（HR）修复，并使用适当设计的外源“供体模板”在断裂附近引入特定突变。最近的原理验证实验表明，ZFNs也可用于在斑马鱼胚胎中产生靶向nhej介导的基因敲除突变。这些新发现提出了一个重要的问题，即如何使工程ZFN技术广泛应用于斑马鱼研究人员，以及ZFN是否可以用于诱导斑马鱼中高效的hr介导的基因靶向。拟议研究的长期目标是提高用于斑马鱼的遗传工具的质量和范围，并使所有斑马鱼研究实验室都能轻松获得定制的ZFN技术。为了实现这些目标，我们将麻省总医院的young和Peterson实验室的锌指工程和斑马鱼专业知识结合起来，提出以下具体目标：(1)使用快速、强大、高效的锌指工程方法OPEN （Oligomerized Pool engineering）设计并公开提供500种不同斑马鱼基因的ZFN对；(2)开发和验证OPEN方法的更快和简化版本（OPEN 2.0版本），以便任何感兴趣的学术实验室都可以使用；(3)证明ZFNs可用于诱导斑马鱼中基于hr的高效基因靶向，并优化该应用的参数。这些目标的成功完成将扩大ZFNs在斑马鱼遗传研究中的使用和应用，斑马鱼是研究人类发育和疾病的既定和重要模式生物。公共卫生相关性：锌指核酸酶（ZFNs）是可定制的“分子剪刀”，可用于改变包括斑马鱼在内的多种生物的特定基因。该提案将寻求使ZFN技术在斑马鱼学术界得到广泛采用，并扩大ZFN可以进行的基因组修饰范围。成功完成本提案的目标将提高用于斑马鱼的遗传工具的质量和范围，斑马鱼是研究人类发育和疾病的重要模式生物。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.