CRISPR-Cas13d: Transgenic zebrafish lines toknockdown mRNA

项目摘要 确定基因的功能是理解脊椎动物发育、调控 机制和人类疾病。基因组编辑技术，如CRISPR-Cas9，允许将 从特定的表型到永久性的基因改变。然而，一些关键的技术和概念问题仍然存在。 这在脊椎动物中是有问题的，特别是在斑马鱼等水生模式生物中。例如，母性的 只要mRNA可以挽救纯合子突变的表型；基因分型步骤繁琐；而长时间的非 编码RNA、致命性或组织/时间基因很难使用DNA操作以及复杂的方法进行研究 基因组基因座。互补性的“击倒”方法是绕过其中一些方法的宝贵工具 然而，问题是，没有系统的工具来敲除斑马鱼或其他硬骨鱼中的mRNA。 我们的长期目标是解开与调节机制、发育和 人类疾病。我们最近的出版物表明，CRISPR-RfxCas13d系统注入到 脊椎动物胚胎提供了一种强大而经济的技术来系统地破坏基因功能。 然而，该系统的注射只提供了~3天的瞬时击倒。因此，中心目标是 该提案的一部分是将CRISPR-RfxCas13d技术从注射到脊椎动物胚胎中转移到 通过转基因获得内源性表达。我们建议开发一个剧目 表达RfxCas13d酶(普遍存在的和组织特异性的)和GuideRNA的转基因斑马鱼将 促进对基因功能的快速和有力的研究。我们的初步数据显示转基因 RfxCas13d在斑马鱼中的表达是功能性的，无毒。目标是：1)定义最优系统 目的：下调CRISPR-RfxCas13d转基因斑马鱼的mRNA表达。2)下调mRNA的表达 以组织特异性的方式使用特定的转基因株系。这项建议在概念上是创新的，因为它是基于 探索以组织特异性方式敲除mRNA的新技术CRISPR-RfxCas13d 在斑马鱼身上。这种方法从未在RNAi不起作用的脊椎动物模型系统中进行过。 该项目的成果将是第一个通过敲门来解剖和研究基因功能的转基因系统。 在斑马鱼中表达下调。我们的方法将帮助科学界研究基因功能 以更快的和组织时间特定的方式，以及回答非常具有挑战性的问题 由当前方法解决的，如非编码RNA的功能或由多个 基因。此外，通过遵循我们将在此项目中优化的指导方针，研究人员可能会产生一个 完整指南RNA收集，针对所有基因，任何人都可以订购以解决这些问题 他们的个人研究问题。最后，由于我们已经成功地实现了CRISPR-Cas13d系统 通过注射其他生物体，如青竹、剑鱼和小鼠胚胎，我们在本提案中描述的工作 可以作为将这种有效的击倒技术转移到一系列其他物种的基础。

Project Summary Determining the function of genes is fundamental for understanding vertebrate development, regulatory mechanisms and human diseases. Genome editing technologies, such CRISPR-Cas9, have allowed associating specific phenotypes to permanent gene alteration. However, some key technical and conceptual issues remain problematic in vertebrates, particularly in aquatic model organisms such zebrafish. For example, the maternally provided mRNA can rescue the phenotype of homozygous mutants; genotyping steps are tedious; and long non- coding RNA, lethal or tissue/temporal genes are difficult to study using DNA manipulation as well as complex genomic loci. Complementary ‘knock-down’ approaches are invaluable tools to circumvent some of these problems, however, there was no systematic tool to knockdown mRNAs in zebrafish or other teleost fish. Our long-term goal is to unravel the function of genes related to regulatory mechanisms, development, and human diseases. Our recent publication demonstrates that injection of the CRISPR-RfxCas13d system into vertebrate embryos provides a robust and cost-effective technology to systematically disrupt gene function. However, the injection of this system only provides transient knockdown for ~3 days. Therefore, the central goal of the proposal is to transfer the CRISPR-RfxCas13d technology from being injected into vertebrate embryos, to being endogenously expressed through transgenesis. We propose that the development of a repertoire transgenic zebrafish expressing the RfxCas13d enzyme (ubiquitous and tissues-specific) and guideRNA would facilitate rapid and vigorous investigation into gene functions. Our preliminary data indicate that transgenic expression of RfxCas13d in zebrafish is functional and not toxic. The objectives are: 1) Define the optimal system to knockdown mRNA expression in CRISPR-RfxCas13d transgenic zebrafish. 2) Knockdown mRNA expression in a tissue-specific manner using specific transgenic lines. This proposal is conceptually innovative as it is based on the exploration of a novel technique, CRISPR-RfxCas13d, to knockdown mRNA in a tissue-specific manner in zebrafish. This approach has never been done in vertebrate model systems where RNAi does not work. The outcomes of this project will be the first transgenic system to dissect and study gene function by knocking down mRNA expression in zebrafish. Our approach will help the scientific community investigate gene function in a faster and tissue-temporal specific manner, as well as answering questions that are very challenging to address by current methodologies, such as the function of non-coding RNAs or phenotypes caused by multiple genes. Moreover, by following the guidelines we will optimize through this project, researchers may produce a whole guideRNA collection, targeting all genes, which should be available for anyone to order to address their individual research questions. Finally, as we have successfully implemented the CRISPR-Cas13d system by injection in other organisms such as medaka, killifish, and mouse embryos, our work described in this proposal may serve as the foundation for transferring this efficient knockdown technology into a range of other species.