ADVANCING GENE-EDITING NUCLEASES FOR DIVERSE ZEBRAFISH APPLICATIONS

CRISPR/Cas9技术的发展改变了我们编辑无数基因组的能力 有机体。今天，几乎任何研究人员都可以实践基本的基因组编辑技术，这要归功于 广泛分布的开放源码CRISPR试剂。尽管如此，该油田的全部潜力还没有得到充分发挥。 进一步的创新可能会带来重大的新进展，使其能够应用于广泛的重要 生物医学问题。未来的几个潜在进展尤其令人兴奋，包括：1)发展 这将使药物发现更有效率，2)，将基因组编辑工具与多样化的 分子技术，以创造新的诊断和生物传感器，以及3)使高效、 全基因组筛选以发现基因功能。这些领域的工作无疑将推动 我们理解、诊断和治疗人类遗传疾病的能力。 在过去的十年里，Peterson、Yeh和Joung实验室之间的合作导致了几个关键的 基因组编辑方面的进展，包括首次使用TALEN编辑斑马鱼基因组，首次使用 CRISPR/Cas9用于修改任何动物的基因组，以及Cas9的第一个工程来改变PAM的特异性。 这些进步总共被引用了数千次，并成为世界各地使用的工具。 我们建议开发三种通过使用基于CRISPR/CAS9的技术来连接的新技术 由于斑马鱼含有多种成分，它们有潜力增加斑马鱼作为模式生物的效用。计划包括： 目的1.建立一种瞬时目视基因分型技术。我们将使用ΦC31DNA 整合酶系统将荧光标记插入到CRISPR/Cas9产生的突变体中，每个突变体具有不同的颜色 基因复制。这将使野生型、杂合型和纯合型的快速鉴定和分类成为可能 来自混合种群的突变，潜在的应用范围从基因研究到药物筛选。 目的2.建立DNA邻近分离报告系统。建立两级联动的记者制度。 CRISPR引导RNA，当靶向彼此附近的两个DNA序列时，将诱导可量化的 发光。该平台的开发将使包括3D地图在内的许多未来应用成为可能 染色体结构和诊断染色体组织缺陷。 目的3.建立斑马鱼高通量CRISPR文库筛选系统。这个 平台将实现sgRNA文库的快速注入以及目标识别标签，随后 对表现出感兴趣表型的动物的选择。致病基因突变的身份将是 通过回收植入的标签而获得。 完成这些目标后，该项目将通过提供以下方面对生物医学研究产生广泛影响 用于定向基因组操作的新工具和方法。

Development of CRISPR/Cas9 technology has transformed our ability to edit the genomes of numerous organisms. Today, almost any investigator can practice the basic genome editing technologies, thanks to the broad distribution of open-source CRISPR reagents. Still, the full potential of the field has not been reached. Further innovation is likely to deliver major new advances, enabling application to a wide range of important biomedical problems. Several potential future advances are particularly exciting, including: 1) developments that will make drug discovery more efficient, 2), developments combining genome editing tools with diverse molecular technologies to create novel diagnostics and biosensors, and 3) developments enabling efficient, genome-wide screens to discover gene functions. Work in these areas will undoubtedly lead to advances in our ability to understand, diagnose, and treat human genetic disorders. Collaboration between the Peterson, Yeh, and Joung labs over the past decade has resulted in several key advances in genome editing, including the first use of TALENs to edit the zebrafish genome, the first use of CRISPR/Cas9 to modify the genome of any animal, and the first engineering of Cas9 to alter PAM specificity. These advances have collectively been cited thousands of times and become tools used around the world. We propose to develop three novel technologies that are connected by their use of CRISPR/Cas9-based components and by their potential to augment the utility of the zebrafish as a model organism. Plans include: Aim 1. To develop a technology for instantaneous visual genotyping. We will use the ΦC31 DNA integrase system to insert fluorescent markers into CRISPR/Cas9-generated mutants, a different color for each gene copy. This will enable rapid identification and sorting of wild-type, heterozygous, and homozygous mutants from a mixed population, with potential applications ranging from genetic studies to drug screening. Aim 2. To create a DNA proximity split-reporter system. We will establish a reporter system in which two CRISPR guide RNAs, when targeted to two DNA sequences located near each other, will induce quantifiable luminescence. Development of this platform will enable numerous future applications including mapping 3D chromosome structure and diagnosing chromosomal organization defects. Aim 3. To develop a system enabling high-throughput CRISPR library screening in zebrafish. The platform will enable rapid injection of libraries of sgRNAs along with target-identifying tags, followed by selection of animals exhibiting phenotypes of interest. The identity of the causative gene disruptions will be obtained by recovery of the implanted tags. Upon completing these aims, this project will have an impact on biomedical research broadly by providing new tools and methodologies for targeted genome manipulation.