Optical Control of Translation and Gene Editing in Zebrafish Embryos

 描述（由申请人提供）：基因表达以时空方式被精确调控和编排，并且多个基因的活性通常相互关联，从而产生高度复杂的遗传网络。基因功能的异常调控可以诱导疾病表型，并与先天性疾病和癌症的发展有关。为了研究基因、基因网络和下游生物学功能，需要以高时空分辨率调节基因表达的能力。光代表理想的调节元件，因为它可以在空间上容易地控制。 和时间的方式，传达生物活动的时空控制的研究中的系统。在解剖脊椎动物基因功能的背景下，斑马鱼已被确立为一个强大的模式生物。已经开发了几种方法来检测斑马鱼胚胎中的基因功能，最常见的方法是通过mRNA注射的基因过表达和通过吗啉代注射的基因沉默。已经报道了斑马鱼胚胎中的光学控制的反义功能;然而，对于光学基因激活或基因编辑，不存在普遍适用的、严格控制的方法。该项目通过开发一种光激活RNA方法来解决这一方法学差距，该方法通过将位点特异性地引入到RNA分子中来直接与已建立的斑马鱼技术相结合。具体而言，将完成两个目标：（1）通过位点特异性引入笼化基团，发展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)开发一种光学控制的CRISPR/Cas9基因组编辑方法，通过位点特异性地将锁定基团引入指导RNA。斑马鱼胚胎中Cas9介导的基因编辑正在迅速成为一种多功能的研究工具，光学控制将为基因组编辑提供前所未有的空间和时间分辨率。所述研究的预期成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。所提出的方法直接接口与完善的RNA转录和注射协议，是标准的斑马鱼社区。斑马鱼胚胎中mRNA和sgRNA功能的紧密、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。

 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.