Development of a platform for spatial functional genomics

项目摘要 基因组中有超过20，000个基因，尽管在分配许多基因方面取得了进展， 尽管我们对基因的功能有了更深入的了解，但我们仍然不知道许多基因的功能或它们在影响疾病中的具体作用。基因 可以指导不同细胞类型、细胞状态或组织中的不同表型，这使得基因注释成为可能。 艰巨的任务需要研究来确定每个基因在不同环境中的功能，但是基因的范围是有限的。 问题是一个挑战-有必要评估100多个基因的许多可能的功能， 一个复杂的细胞和细胞外环境的背景下，其空间结构是一个重要组成部分， 系统行为此外，一些基因与其他基因协调或冗余地工作，使表型 注释更具挑战性。确定每个基因在不同的正常和疾病状态下的作用， 对生物学和医学产生了不可估量的影响。它会引出不同的新的内在成分， 因此，它有助于开发治疗这些疾病的药物。 该项目的目标是建立一个空间功能基因组学的第一个平台（扰动- map），这将使包括多基因敲除在内的数百种遗传干扰并行产生 在组织和肿瘤中，以及每种扰动对多种内在和外在生物过程的影响 以细胞、亚细胞和组织水平分辨率显示。为了达到我们的目标，我们将开发新的 技术，方法和软件工具，将允许数以百计的CRISPR，沿着几十个生物学 表型，通过高维成像在组织内空间分辨。为了确定 扰动地图，以解决迫切的未满足的需求，我们将应用该平台的最紧迫的问题之一， 免疫肿瘤学，即肿瘤如何阻止免疫浸润和破坏免疫。我们将使用扰动- 图谱以确定调节免疫募集和从肿瘤微环境中排除的因素， 发现控制肿瘤对免疫清除抵抗的基因。 该项目的成果将是空间细胞功能基因组学的变革性技术， 以及亚细胞分辨率和高维表型。这将具有无与伦比的能力， 在广泛的生物学领域回答许多问题，并调查整个类别的基因， 无法用现有功能基因组学方法研究的表型; 研究确定控制过程的基因，如：组织和肿瘤组织，细胞迁移，侵袭 和转移、细胞-细胞相互作用以及局部免疫细胞募集和串扰。通过能够 在同一个细胞内询问多个基因扰动，扰动图也将使遗传冗余 和合成致死关系有待确定，这是靶向癌症发展的一个主要领域。 治疗因此，该平台将在基础和转化生物学的许多领域具有广泛的实用性。

PROJECT SUMMARY There are >20,000 genes in the genome and though there has been progress in assigning many gene functions, we still do not know the function of numerous genes or their specific roles in affecting disease. Genes can direct diverse phenotypes in different cell types, cell states, or tissues, which makes gene annotation a staggering task. Studies are needed to determine each gene’s function in different contexts, but the scope of the problem is a challenge – it is necessary to assess many possible functions of 100s of genes and within the context of a complex cellular and extracellular milieu whose spatial architecture is an essential component of system behavior. In addition, some genes work coordinately or redundantly with other genes, making phenotypic annotation more challenging. Establishing the role of every gene in different normal and disease states would have an incalculable impact on biology and medicine. It would elicit novel intrinsic components of different diseases and thus facilitate the development of drugs to treat some of these diseases. The objective of this project is to establish a first-of-its-kind platform for spatial functional genomics (Perturb- map), which will enable 100s of genetic perturbations, including multi-gene knockouts, to be generated in parallel in tissues and tumors, and the effect of each perturbation on multiple intrinsic and extrinsic biological processes to be revealed at cellular, subcellular, and tissue level resolution. To reach our objective, we will develop novel technologies, methods, and software tools that will permit 100s of CRISPRs, along with dozens of biological phenotypes, to be spatially resolved within a tissue by high-dimensional imaging. To establish the potential of Perturb-map to address urgent unmet needs, we will apply the platform to one of the most pressing questions in immune oncology, namely how tumors prevent immune infiltration and subvert immunity. We will use Perturb- map to identify factors that regulate immune recruitment and exclusion from the tumor microenvironment and uncover genes controlling tumor resistance to immune clearance. The outcome of this project will be a transformative technology for functional genomics with spatial cellular and sub-cellular resolution and high-dimensional phenotyping. This would have unmatched capabilities to answer numerous questions in a broad array of biological areas, and to investigate entire classes of genes and phenotypes that could not be studied with existing functional genomics approaches; enabling highly scaled studies to identify genes that control processes such as: tissue and tumor organization, cell migration, invasion and metastasis, cell-cell interactions, and local immune cell recruitment and crosstalk. By being able to interrogate multiple gene perturbations within the same cell, Perturb-map will also enable genetic redundancies and synthetic lethal relationships to be identified, a major area of interest for development of targeted cancer therapies. Thus, the platform will have broad utility in numerous areas of basic and translational biology.