ADVANCING GENE-EDITING NUCLEASES FOR TARGETED GENOME MANIPULATION

描述(申请人提供)：锌指核酸酶(ZFN)和转录激活物样效应核酸酶(TALEN)的快速发展改变了我们在斑马鱼中进行靶向基因敲除的能力。制造ZFN或TALEN介导的基因敲除的工具和技术正在变得非常精致和广泛分布。在过去三年里，在这笔赠款的支持下，开发了用于工程ZFN和TALEN的公开可用方案、试剂和软件，并为大量内源斑马鱼基因生成了锌指阵列和TALEN。因此，今天几乎任何斑马鱼研究人员都可以利用这些资源敲除他们感兴趣的基因。尽管基因敲除对于了解基因功能很重要，但零等位基因只是斑马鱼生物学研究的一种工具。为了全面了解基因的功能，在空间和时间上控制基因表达的方法将提供强大的工具。基因工程的锌指和转录激活因子样效应子重复DNA结合域有可能对所有这些区域做出贡献，从而进一步增强斑马鱼作为实验模型的实用性。在这一更新应用中，我们建议继续我们高效的合作努力，并超越常规的基因敲除，探索在斑马鱼中工程DNA结合域的其他新应用。在项目期间，将开发TALES的几种新应用，包括创建人工转录因子，构建一套TALENS THA将能够破坏所有斑马鱼的microRNA，以及产生几种不同类型的 条件等位基因。具体目标是：目标1：开发调控斑马鱼内源基因表达的基于TALE的转录因子。目的2：产生一套针对所有斑马鱼microRNAs(MiRNAs)的全套TALEN。目的3：开发能够有条件地改变基因结构和表达的新工具。通过完成这些目标，我们将推进基因组编辑领域，并大幅提高斑马鱼作为实验模型的实用性。根据我们对“开源”科学的坚定和可靠的承诺，我们将继续向学术研究人员公开提供实践本提案中开发的技术所需的所有试剂、协议和软件。我们相信，这项提案的研究团队在共同工作的生产力以及开发对社区广泛有用的工具和试剂方面有着良好的记录。

DESCRIPTION (provided by applicant): Rapid advances with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have transformed our ability to make targeted gene knockouts in zebrafish. Tools and techniques for making ZFN- or TALEN-mediated knockouts are becoming well refined and broadly distributed. Efforts supported by this grant over the previous three years have resulted in the development of publicly available protocols, reagents, and software for engineering ZFNs and TALENs and have generated zinc finger arrays and TALENs for a large set of endogenous zebrafish genes. As a result, today almost any zebrafish investigator can knock out their genes of interest using these resources. Despite the importance of gene knockouts for understanding gene function, null alleles represent only one tool for biological research in zebrafish. To understand gene function completely, methods for controlling gene expression both spatially and temporally would provide powerful tools. Engineered zinc finger and transcription activator-like effector repeat DNA-binding domains have the potential to contribute to all of these areas and to thereby further enhance the utility of the zebrafish as an experimental model. In this renewal application, we propose to continue our highly productive collaborative efforts and to move beyond routine gene knockouts to explore additional novel applications for engineered DNA-binding domains in the zebrafish. During the project period, several novel applications for TALEs will be developed, including the creation of artificial transcription factors, the construction of a set of TALENs tha will enable disruption of all zebrafish microRNAs, and the generation of several different types of conditional alleles. The specific aims are: Aim 1: To develop TALE-based transcription factors for controlling endogenous gene expression in zebrafish. Aim 2: To generate a comprehensive set of TALENs targeted to all zebrafish microRNAs (miRNAs). Aim 3: To develop novel tools that enable conditional alteration of gene structure and expression. By completing these aims, we will advance the field of genome editing and substantially enhance the utility of the zebrafish as an experimental model. Consistent with our strong and well-established commitment to "open-source" science, we will continue to make all of the reagents, protocols, and software needed to practice the technologies developed in this proposal publicly available to academic researchers. We believe that the team of researchers for this proposal has a strong track record of productivity working together and of generating tools and reagents that will be broadly useful to the community.