ADVANCING GENE-EDITING NUCLEASES FOR DIVERSE ZEBRAFISH APPLICATIONS

推进基因编辑核酸酶在多种斑马鱼中的应用

基本信息

批准号：
10470200
负责人：
RANDALL T PETERSON
金额：
$ 47.04万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-20 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10470200
关键词：
3-Dimensional Animal Model Animals Area Biological Assay Biomedical Research Biosensor CRISPR library CRISPR screen CRISPR/Cas technology Cells Chromosomal translocation Chromosome Structures Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Color DNA DNA Sequence Defect Development Diagnosis Disease Drug Screening Engineering Exhibits Future Generations Genes Genetic Diseases Genetic Screening Genetic study Genome Genotype Guide RNA Heterozygote Homozygote Human Genetics Implant Injections Integrase Lead Libraries Methodology Microfluidics Molecular Mutation Mutation Detection Needles Organism Output Phenotype Population Reagent Recovery Reporter Research Personnel Science Sorting - Cell Movement Specificity System Techniques Technology Therapeutic Agents Time Toxin Visual Work Zebrafish base chemical genetics drug discovery functional genomics gene discovery gene function genome editing genome wide screen genome-wide in vivo innovation interest luminescence mutant new technology novel novel diagnostics nuclease open source rapid detection reconstitution reverse genetics screening tool transcription activator-like effector nucleases zebrafish genome zinc finger nuclease zinc finger nuclease technology

项目摘要

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.

CRISPR/Cas9 技术的发展改变了我们编辑众多基因组的能力今天，几乎所有研究人员都可以练习基本的基因组编辑技术，这要归功于开源 CRISPR 试剂的广泛分布尽管如此，该领域的潜力尚未充分发挥。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领域一些潜在的未来进展特别令人兴奋，包括：1）发展。这将使药物发现更加高效，2）将基因组编辑工具与多样化相结合的发展分子技术来创建新型诊断和生物传感器，以及 3) 实现高效、通过全基因组筛选来发现基因功能，这些领域的工作无疑将带来进步。我们理解、诊断和治疗人类遗传疾病的能力。 Peterson、Yeh 和 Joung 实验室在过去十年中的合作取得了多项关键成果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 可修改任何动物的基因组，并且是第一个改变 PAM 特异性的 Cas9 工程。这些进步已被引用数千次，并成为世界各地使用的工具。我们建议开发三项新技术，通过使用基于 CRISPR/Cas9 的技术将它们联系起来组成部分及其增强斑马鱼作为模式生物的效用的潜力。计划包括：目标 1. 开发一种即时视觉基因分型技术我们将使用 ΦC31 DNA。整合酶系统将荧光标记插入 CRISPR/Cas9 生成的突变体中，每个突变体有不同的颜色这将能够快速识别和分选野生型、杂合子和纯合子。来自混合群体的突变体，潜在应用范围从遗传研究到药物筛选。目标 2. 创建 DNA 邻近分裂报告系统我们将建立一个包含两个的报告系统。当 CRISPR 引导 RNA 靶向彼此靠近的两个 DNA 序列时，将诱导可量化的该平台的开发将使许多未来应用成为可能，包括 3D 地图绘制。染色体结构和诊断染色体组织缺陷。目标 3. 开发一种能够在斑马鱼中进行高通量 CRISPR 文库筛选的系统。平台将能够快速注入 sgRNA 文库以及目标识别标签，然后选择表现出感兴趣的表型的动物将是致病基因破坏的身份。通过恢复植入的标签获得。完成这些目标后，该项目将通过提供以下内容对生物医学研究产生广泛影响：用于靶向基因组操作的新工具和方法。