ADVANCING ZINC FINGER NUCLEASES FOR TARGETED GENOME MANIPULATION

推进锌指核酸酶的靶向基因组操作

• 批准号: 8303297
• 负责人: J. KEITH JOUNG
• 金额: $ 56.36万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8303297
• 关键词: 3' -nuclease; Adoption; Animal Model; Cells; Communities; Cystic Fibrosis Transmembrane Conductance Regulator; DNA Double Strand Break; DNA Sequence; Drosophila genus; Embryo; Engineering; Gene Targeting; General Hospitals; Genes; Genetic; Genome; Goals; Health; Human; Human Development; Knock-out; Laboratories; Laboratory Research; Massachusetts; Mediating; Methods; Modification; Molecular; Mutation; Nonhomologous DNA End Joining; Organism; Plant Genes; Plants; Process; Recording of previous events; Reporter Genes; Reporting; Research; Research Personnel; Scientist; Site; Technology; Telomerase; Tobacco; Vascular Endothelial Growth Factors; Zebrafish; Zinc Fingers; base; design; dopamine transporter; experience; homologous recombination; human disease; improved; insertion/deletion mutation; interest; nuclease; repaired; research study; restriction enzyme; scale up; tool

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.

描述（由申请人提供）：锌指核酸酶（ZFN）是可定制的核酸酶，用于改变果蝇，植物和人类细胞的内源基因，其效率高1％-50％。 ZFN诱导的DNA双链断裂（DSB）可以通过容易出错的非同源最终连接（NHEJ）或具有适当设计的外源性“供体模板”的同源重组（HR）来修复，以在断裂附近引入特异性突变。最新的原则实验表明，ZFN也可用于在斑马鱼胚胎中创建靶向的NHEJ介导的敲除突变。这些新发现提出了有关如何使斑马鱼研究人员广泛使用的工程ZFN技术以及是否可以使用ZFN来诱导斑马鱼中高效的HR介导的基因靶向的重要问题。拟议的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可以轻松访问定制的ZFN技术。 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）开发和验证开放方法的更快，更简化的版本（Open 2.0版），以便任何感兴趣的学术实验室都可以使用它； （3）证明ZFN可用于在斑马鱼中诱导高效的基于HR的基因靶向并优化此应用程序的参数。这些目标的成功完成将扩大ZFN在斑马鱼的遗传研究中的使用和应用，斑马鱼是一种研究人类发育和疾病的既定且重要的模型生物。公共卫生相关性：锌指核酸酶（ZFN）是可自定义的“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。该建议将旨在通过学术斑马鱼社区对ZFN技术进行广泛采用，并扩大可以使用ZFN进行的基因组修改范围。成功完成该提案的目的将提高可用于研究人类发育和疾病的重要模型生物的遗传工具的质量和范围。