ADVANCING GENE-EDITING NUCLEASES FOR TARGETED GENOME MANIPULATION

描述（由申请人提供）：锌指核酸酶（ZFNs）和转录激活物样效应核酸酶（TALENs）的快速进展已经改变了我们在斑马鱼中进行靶向基因敲除的能力。用于制造ZFN或talen介导的敲除的工具和技术正变得越来越完善和广泛分布。在过去的三年里，该基金支持的工作导致了ZFNs和TALENs工程的公开可用协议、试剂和软件的发展，并为大量内源性斑马鱼基因生成了锌指阵列和TALENs。因此，今天几乎任何斑马鱼研究者都可以利用这些资源敲除它们感兴趣的基因。尽管基因敲除对了解基因功能很重要，但无效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，从空间和时间上控制基因表达的方法将提供有力的工具。工程锌指和转录激活因子样效应重复dna结合域有潜力为所有这些领域做出贡献，从而进一步提高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，探索工程dna结合域在斑马鱼中的其他新应用。在项目期间，TALEs的几个新应用将被开发，包括人工转录因子的创建，一组TALENs的构建，可以破坏所有斑马鱼的microrna，以及几种不同类型的dna的生成

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.