Orthogonal CRISPR GEMMs

正交 CRISPR GEMM

基本信息

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
项目类别：
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

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

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项目摘要

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.

项目摘要该提议的核心是推翻研究人类的现有单基因范式有机模型研究中的基因，并推动在体内研究遗传相互作用的包膜。我们已经开发了一种技术，可以使用高通量来研究小鼠模型中的基因相互作用 CRISPR技术适用于在给定途径或疾病中实施的特定基因。从未在任何人中完成一种体内高通量针对目标的多突击方法有机模型，这将彻底改变物理上复杂基因相互作用的研究相关的有机模型系统。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平台并应用严格/可重复性。多聚机技术将有助于弥合由基因组测序研究以及使用这些数据来理解生物学和疾病。我们的最终目标是对提出的技术进行基准测试，以说明其在用例中的应用设置 - 用正交基因激活和基因编辑CRISPR键入一组CD抗原揭示基本遗传相互作用和途径方向性的机械。我们的一般策略是利用过去两年中我们开发的新型工具和方法 - 使用创新的高吞吐量CRISPR筛选方法。我们的最终目标是开发一个模块化推进功能基因组学方法的工具集。所有这些都将在动物模型中在体内完成。我们的目标是试行一个正交的多聚机平台，以调查多达900个组合单个动物的体内扰动。我们将使用CD抗原作为我们的技术作为使用市售单克隆抗体易于定量的报告基因。有针对性的编辑和笔录丰度将通过流式细胞术和单细胞分析对B和T细胞的亚群进行测序。复杂疾病的生物信息剖析机制的未来是有希望的，但挑战。人类序列的多个大规模参考数据集正在迅速成为可用，预计将在未来几十年中增加。数百万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，没有可行的方法来询问成千上万的组合动物模型中的基因。该提案旨在进一步推进将提高复杂的新技术遗传学问题与有机生物学和人类疾病有关，并将展示有望新的研究体内遗传相互作用的技术。