ADVANCING ZINC FINGER NUCLEASES FOR TARGETED GENOME MANIPULATION

描述(申请人提供)：锌指核酸酶(ZFN)是一种可定制的核酸酶，用于改变果蝇、植物和人类细胞的内源基因，效率从1%-50%不等。ZFN诱导的DNA双链断裂(DSB)可以通过容易出错的非同源末端连接(NHEJ)或通过与适当设计的外源“供体模板”进行同源重组(HR)来修复，以在断裂附近引入特定的突变。最近的原理验证实验表明，ZFN也可以用来在斑马鱼胚胎中创建NHEJ介导的靶向基因敲除突变。这些新的发现提出了重要的问题，即如何使工程ZFN技术广泛应用于斑马鱼研究人员，以及ZFN是否可以用于在斑马鱼中诱导高效的HR介导的基因靶向。拟议研究的长期目标是提高可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容易获得定制的ZFN技术。为了实现这些目标，我们正在结合马萨诸塞州总医院Joung和Peterson实验室的锌指工程和斑马鱼专业知识，提出以下具体目标：(1)使用一种称为OPEN的快速、健壮和高效的锌指工程方法设计并公开提供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版本2.0)，以便它可以被任何感兴趣的学术实验室使用；(3)证明ZFN可以在斑马鱼中诱导高效的基于HR的基因打靶，并优化这一应用的参数。这些目标的成功实现将扩大ZFN在斑马鱼遗传研究中的用途和应用，斑马鱼是研究人类发育和疾病的一种已建立的重要模式生物。与公共健康相关：锌指核酸酶(ZFN)是一种可定制的“分子剪刀”，可用于改变包括斑马鱼在内的各种生物中的特定基因。这项提议将寻求使ZFN技术在学术上得到斑马鱼界的广泛采用，并扩大ZFN可以进行的基因组修改的范围。成功完成这项提议的目标将提高可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.