A germline- and promoter-independent strategy to gain access to all cell types in the brain

一种独立于种系和启动子的策略，可获取大脑中所有细胞类型

• 批准号: 10651435
• 负责人: HUAIYU HU
• 金额: $ 341.87万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10651435
• 关键词: 3' Untranslated Regions; Amaze; Anatomy; Animal Model; Attention; Brain; CRISPR/Cas technology; Callithrix; Capsid; Cell physiology; Cre driver; Cre lox recombination system; DNA; DNA sequencing; Dependovirus; Development; Electrophysiology (science); Enterobacteria phage P1 Cre recombinase; Fibroblasts; Gene Expression; Genes; Genetically Modified Animals; Goals; Guide RNA; Heterogeneity; High-Throughput Nucleotide Sequencing; Immune response; Immunohistochemistry; In Vitro; Internal Ribosome Entry Site; Knowledge; Libraries; Mediating; Methods; Modification; Molecular; Mus; Neurons; Nonhomologous DNA End Joining; Population; Primates; Process; Property; Reporter; Research; Specificity; System; Technology; Testing; Transgenic Animals; Vision; Visual Cortex; Work; brain cell; cell type; cost; design; experimental study; genome editing; improved; in vivo; mRNA delivery; multiplex assay; nervous system disorder; neural circuit; neuroinflammation; nonhuman primate; novel; promoter; recombinase; technology development; tool; transcriptome

Project summary The heterogeneity from the vast number of cell types in the brain presents a major challenge in our understanding of how brain works and in our treatment of neurological disorders. With the amazing advances in high throughput sequencing technology, our knowledge on the molecular makeup of the myriad cell types in the brain has reached an unprecedented level. However, tools that allow us to easily study the functions of any cell types of our choice are lagging. The goal of our proposed research is to develop technology to generate such tools. In order to target a specific cell type, current approaches typically depend on genetically modified animal models, which is laborious, costly, and low throughput, or well-defined and small cell-type-specific promoters, which still remain to be difficult to isolate. Here we propose to develop an AAV-based Cell-Type-Specific Targeting (ACTSTar) system to enable easy access to any cell type in the brain in any species that is independent of germline modification and promoter isolation and characterization. We plan to generate a library of ACTSTar drivers to target 50 different brain cell types in the mouse and marmoset. In addition, to reduce off-target integration and neuroinflammation associated with long-term Cas9 expression, especially in long-term experiments in primates, we will develop a novel AAV-capsid mediated Cas9 mRNA delivery system for transient Cas9 expression. This project will generate the much-needed tools for easy access to any brain cell types in multiple species. Our research goal will be facilitated by the complementary expertise of the MPI team in genome editing and developing safe Cas9 delivery methods (Lu), neural circuit functions of the mouse brain (Lin), and visual functions using non-human primates (Hu).

项目摘要 大脑中大量细胞类型的异质性在我们理解中提出了一个主要挑战 关于大脑的工作方式以及我们对神经系统疾病的治疗。随着高吞吐量的惊人进步 测序技术，我们对大脑中无数细胞类型的分子构成的了解 达到了空前的水平。但是，使我们可以轻松研究任何单元类型的功能的工具 我们的选择滞后。我们拟议的研究的目的是开发技术来生成此类工具。在 为了靶向特定的细胞类型，电流方法通常取决于转基因动物模型， 这是费力的，昂贵的，低吞吐量的，或定义明确的小细胞类型的启动子，仍然 仍然很难分离。在这里，我们建议开发基于AAV的细胞类型特异性靶向 （ACTSTAR）系统可以轻松访问任何独立于任何物种的大脑中的任何细胞类型 种系修饰和启动子的隔离和表征。我们计划生成Actstar库 驱动器靶向小鼠和果棒中的50种不同的脑细胞类型。此外，要减少脱靶 与长期CAS9表达相关的整合和神经炎症，尤其是在长期中 灵长类动物的实验，我们将开发一种新型的AAV capsid介导的Cas9 mRNA输送系统，以进行瞬态 CAS9表达。该项目将生成急需的工具，以方便访问任何脑细胞类型 多种物种。 MPI团队在基因组中的补充专业知识将促进我们的研究目标 编辑和开发安全的CAS9输送方法（LU），小鼠大脑的神经回路功能（LIN）和 使用非人类灵长类动物（HU）的视觉功能。