Optical Control of Translation and Gene Editing in Zebrafish Embryos

斑马鱼胚胎翻译和基因编辑的光学控制

• 批准号: 9357624
• 负责人: Alexander Deiters
• 金额: $ 19.46万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-23 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9357624
• 关键词: Address; Animal Model; Binding; Biological; Biological Models; Biological Process; CRISPR/Cas technology; Cell Culture Techniques; Cells; Communities; Complex; Congenital Disorders; DNA; Development; DsRed; Embryo; Event; Funding; Future; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genetic Transcription; Grant; Guide RNA; In Vitro; Initiator Codon; Injection of therapeutic agent; Investigation; Knowledge; Lead; Left; Length; Ligands; Light; Link; Malignant Neoplasms; Mediating; Messenger RNA; Methodology; Methods; Molecular; Nature; Oligonucleotides; Optical Methods; Optics; Outcome; Pattern; Protocols documentation; RNA; Regulatory Element; Reporter; Reporting; Research; Research Personnel; Resolution; Site; System; Techniques; Testing; Transforming Growth Factor beta; Translations; Zebrafish; base; disease phenotype; experience; gene function; genome editing; nucleobase; overexpression; phosphoramidite; photolysis; prevent; public health relevance; spatiotemporal; stability testing; temporal measurement; tool

 DESCRIPTION (provided by applicant): Gene expression is precisely regulated and orchestrated in a spatio-temporal fashion and the activity of multiple genes is often interlinked, creating highly complex genetic networks. The aberrant regulation of gene function can induce disease phenotypes and has been connected with congenital disorders and cancer development. In order to investigate genes, gene networks, and downstream biological functions, the ability to regulate gene expression with high spatio-temporal resolution is desirable. Light represents an ideal regulatory element as it can be easily controlled in a spatial and a temporal fashion, conveying spatio-temporal control of biological activity to the system under study. In the context of dissecting vertebrate gene function, the zebrafish has been established as a powerful model organism. Several methods have been developed for examining gene function in zebrafish embryos, the most common ones being gene overexpression through mRNA injection and gene silencing through morpholino injection. Optically controlled antisense function in zebrafish embryos has been reported; however, no generally applicable, tightly controlled methodology exists for optical gene activation or gene editing. This project addresses this methodology-gap by developing a light-activated RNA approach that directly interfaces with established zebrafish techniques by site-specifically introducing caging groups into RNA molecules. Specifically, two aims will be completed: (1) Develop an mRNA light-activation methodology through the site- specific introduction of caging groups, in order to achieve rapid and precisely regulated gene activation in zebrafish embryos. (2) Develop an optically controlled CRISPR/Cas9 genome editing methodology through the site-specific introduction of caging groups into guide RNA. Cas9-mediated gene editing in zebrafish embryos is rapidly emerging as a versatile research tool and optical control will provide unprecedented spatial and temporal resolution over genome editing. The expected outcomes of the described research are general methods for the spatio-temporal control of gene function through optical activation of gene expression and gene editing in zebrafish embryos. The proposed methodologies directly interface with well-established RNA transcription and injection protocols that are standard in the zebrafish community. The development of tight, non-leaky, and fully predictable optical control of mRNA and sgRNA function in zebrafish embryos will enable a plethora of biological investigations that will have a long-term impact on the field and will be the enabling methodologies for a wide range of future studies.

 描述(申请人提供)：基因表达是以时空方式精确调控和编排的，多个基因的活动往往是相互关联的，形成了高度复杂的遗传网络。基因功能的异常调节可以诱发疾病表型，并与先天性疾病和癌症的发生有关。为了研究基因、基因网络和下游生物功能，需要具有高时空分辨率的基因表达调控能力。光是一种理想的调节元素，因为它可以很容易地在空间中进行控制 以及一种时间时尚，将对生物活动的时空控制传递给正在研究的系统。在解剖脊椎动物基因功能的背景下，斑马鱼已被确立为一种强大的模式生物。检测斑马鱼胚胎基因功能的方法有几种，最常见的方法是通过注射mRNA实现基因过表达和通过注射吗啡实现基因沉默。已有关于斑马鱼胚胎中光学控制反义功能的报道；然而，对于光学基因激活或基因编辑，还没有普遍适用的、严格控制的方法。该项目通过开发一种光激活的RNA方法来解决这一方法学空白，该方法通过将笼状基团特异性地引入到RNA分子中，直接与已有的斑马鱼技术对接。具体地说，将完成两个目标：(1)通过定点引入笼状基团，建立一种mRNA光激活方法，以实现斑马鱼胚胎中快速、精确调控的基因激活。(2)通过在GUIDE RNA中特异性地引入笼状基团，建立一种光控CRISPR/Cas9基因组编辑方法。Cas9介导的斑马鱼胚胎基因编辑正迅速成为一种通用的研究工具，光学控制将在基因组编辑方面提供前所未有的空间和时间分辨率。所述研究的预期结果是通过斑马鱼胚胎中基因表达的光学激活和基因编辑来时空控制基因功能的一般方法。建议的方法直接与斑马鱼群落中标准的成熟的RNA转录和注射协议相结合。对斑马鱼胚胎中的mRNA和sgRNA功能进行严密、无泄漏和完全可预测的光学控制的发展将使大量的生物学研究成为可能，这些研究将对该领域产生长期影响，并将成为未来广泛研究的方法。