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

• 批准号: 10245123
• 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
• 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/10245123
• 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

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技术的发展已经改变了我们编辑许多人类基因组的能力。 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 开源CRISPR试剂的广泛分布。然而，该领域的全部潜力尚未得到发挥。 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。 生物医学问题。几个潜在的未来进展特别令人兴奋，包括：1）发展 这将使药物发现更有效，2）将基因组编辑工具与多种 分子技术，以创造新的诊断和生物传感器，和3）发展， 全基因组筛选以发现基因功能。这些领域的工作无疑将促进 我们理解、诊断和治疗人类遗传疾病的能力。 Peterson、Yeh和Jorge实验室在过去十年中的合作已经产生了几个关键的成果。 基因组编辑的进展，包括首次使用TALENs编辑斑马鱼基因组，首次使用 CRISPR/Cas9修饰任何动物的基因组，以及Cas9的第一次工程改造以改变PAM特异性。 这些进步被引用了数千次，并成为世界各地使用的工具。 我们建议开发三种新技术，这些技术通过使用基于CRISPR/Cas9的 它们的潜力，以增加斑马鱼作为模式生物的实用性。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产生的突变体中，每个突变体的颜色不同 基因拷贝这将使得能够快速鉴定和分选野生型、杂合型和纯合型。 突变体从混合人口，具有潜在的应用范围从遗传研究到药物筛选。 目标二。创造一个DNA邻近分裂报告系统。我们将建立一个记者制度，其中两个 当CRISPR向导RNA靶向位于彼此附近的两个DNA序列时，将诱导可定量的CRISPR。 发光该平台的开发将使许多未来的应用程序，包括地图3D 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。的 该平台将能够快速注射sgRNA文库沿着靶标识别标签，然后 选择表现出感兴趣的表型的动物。致病基因破坏的身份将是 通过回收植入的标签获得。 在完成这些目标后，该项目将通过提供生物医学研究的广泛影响， 用于靶向基因组操作的新工具和方法。