Orthogonal CRISPR GEMMs

正交 CRISPR GEMM

• 批准号: 10639698
• 负责人: MICHAEL T MCMANUS
• 金额: $ 66.93万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10639698
• 关键词: Address; Adoption; Alleles; Animal Model; Animals; Antigen Targeting; B-Lymphocytes; Benchmarking; Bioinformatics; Biological Models; Biology; CD Antigens; CRISPR screen; CRISPR-mediated transcriptional activation; CRISPR/Cas technology; Cell Line; Cells; Chromosome Mapping; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; DNA Sequence Alteration; Data; Data Set; Development; Diabetes Mellitus; Disease; Etiology; Flow Cytometry; Future; Gene Activation; Gene Combinations; Genes; Genetic; Genetic Epistasis; Genetic Recombination; Genetic study; Genetically Engineered Mouse; Genomic approach; Genomics; Goals; Guide RNA; Heart; Human; Libraries; Malignant Neoplasms; Methodology; Methods; Modality; Modeling; Molecular Profiling; Monoclonal Antibodies; Mus; Mutation; Pathway interactions; Performance; Phenotype; Physiological; Publishing; RNA library; Reagent; Reporter Genes; Reproducibility; Research; Resources; Study models; T-Lymphocyte; Technology; Testing; Time; Transcript; Transgenic Mice; Validation; Work; experimental study; functional genomics; gene interaction; genetic variant; genome sequencing; human disease; human genome sequencing; in vivo; innovation; insertion/deletion mutation; mouse model; mutant; nervous system disorder; new technology; next generation; novel; prime editor; programs; single cell sequencing; technology validation; tool

PROJECT SUMMARY The heart of this proposal is to overturn the existing one-gene-at-a-time paradigm for studying human genes in organismal model studies, and to push the envelope for studying genetic interactions in vivo. We have developed a technology to study gene interactions in mouse models using a high-throughput CRISPR technology suitable for interrogating specific genes implicated in a given pathway or disease. An in vivo high-throughput targeted multi-mutation approach has never been accomplished in any organismal model and this will revolutionize the study of complex gene interaction in physiologically relevant organismal model systems. Our preliminary data have addressed the major feasibility gaps but we need to further develop the platform and apply rigor/reproducibility. Multimer technology will help bridge the gap between the enormous volumes of data generated by genome sequencing studies and the ability to use these data for the understanding of biology and disease. Our end goal is to benchmark the proposed technology, illustrating its application in a use-case setting—targeting a set of CD antigens with orthogonal gene activation and gene editing CRISPR machinery to reveal underlying genetic interactions and pathway directionality. Our general strategy is to take advantage of novel tools and methodologies that we have developed during the past two years– using innovative high throughput CRISPR screening methods. Our end goal is to develop a modular toolset that advances functional genomics approaches. All this will be done in vivo in an animal model. Our goal is to pilot an orthogonal Multimer platform to investigate up to 900 combinations of perturbations in vivo in a single animal. We will benchmark our technology using CD antigens as reporter genes that are easy to quantitate using commercially available monoclonal antibodies. Targeted edits and transcript abundance will be analyzed by flow cytometry and via single-cell sequencing on subpopulations of B and T cells. The future for bioinformatically dissecting mechanisms of complex diseases is promising but challenging. Multiple large-scale reference data sets of human sequences are rapidly becoming available and are expected to increase over the coming decades. Millions of human genome sequencing data sets will constitute an incredible resource for interpretation of DNA mutations. Unfortunately, there are no feasible approaches for interrogating the thousands of combinations of genes in animal models. This proposal aims to further a new technology that would advance complex genetics problems relevant to organismal biology and human disease and will showcase promising new technologies for studying genetic interaction in vivo.

项目总结 这一提议的核心是颠覆现有的一次一个基因研究人类的范式 生物模型研究中的基因，并推动研究体内遗传相互作用的极限。 我们已经开发了一种技术来研究小鼠模型中的基因交互作用，使用高通量 CRISPR技术适用于询问与特定途径或疾病有关的特定基因。 体内高通量靶向多突变方法从未在任何 生物体模型，这将彻底改变生理学中复杂基因相互作用的研究。 相关的生物模型系统。我们的初步数据已经解决了主要的可行性差距，但 我们需要进一步开发该平台，并应用严格性/重复性。 多计时器技术将有助于弥合以下数据之间的差距 基因组测序研究以及利用这些数据了解生物学和 疾病。我们的最终目标是对提议的技术进行基准测试，并说明其在用例中的应用 设置-靶向一组具有正交基因激活和基因编辑CRISPR的CD抗原 揭示潜在的遗传相互作用和途径方向性的机制。我们的总体战略是 利用我们在过去两年开发的新工具和方法- 使用创新的高通量CRISPR筛查方法。我们的最终目标是开发模块化的 推进功能基因组学方法的工具包。所有这些都将在活体动物模型中完成。 我们的目标是试行一个正交多点平台，以调查多达900种组合 单个动物体内的微扰。我们将使用CD抗原作为我们的技术基准 报告基因很容易使用商业上可获得的单抗进行定量。 目标编辑和转录丰度将通过流式细胞仪和单细胞进行分析 B细胞和T细胞亚群的测序。 复杂疾病的生物信息解剖机制的前景是光明的，但 很有挑战性。人类序列的多个大规模参考数据集正在迅速成为 可供使用，预计在未来几十年将会增加。数百万个人类基因组 测序数据集将构成解释DNA突变的令人难以置信的资源。 不幸的是，没有可行的方法来审问数以千计的 动物模型中的基因。这项提议旨在推动一项新技术的发展，以推动复杂的 与生物生物学和人类疾病相关的遗传学问题，并将展示有前景的新 研究体内遗传相互作用的技术。