A new approach to biological recording of lineage hierarchy in primate brains

灵长类大脑谱系层次生物记录的新方法

• 批准号: 9795184
• 负责人: ALI H BRIVANLOU
• 金额: $ 120.77万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-12 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9795184
• 关键词: 3-Dimensional; Animal Model; Animals; Biological; Bioreactors; Brain; Bypass; CRISPR/Cas technology; Callithrix; Cells; Cicatrix; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complex; Controlled Environment; Data; Data Analyses; Development; Developmental Biology; Dimensions; Disease; Embryo; Event; Evolution; Generations; Genetic; Guide RNA; Heterogeneity; Human; Human Development; Immunology; In Vitro; Inherited; Injections; Interneurons; Knowledge; Life Cycle Stages; Link; Malignant Neoplasms; Mammals; Molecular; Neuroglia; Neurons; Neurosciences; Nucleotides; Occipital lobe; Organ; Organism; Organoids; Output; Phylogenetic Analysis; Primates; Process; Property; Radial; Resolution; Seeds; Stem cells; Subfamily lentivirinae; System; Techniques; Technology; Time; Tissues; Totipotent; Trees; Variant; Whole Organism; base; cell type; comparative; daughter cell; experimental study; frontal lobe; functional restoration; human embryonic stem cell; in utero; in vivo; induced pluripotent stem cell; nerve stem cell; new technology; nonhuman primate; novel; novel strategies; progenitor; reconstruction; single-cell RNA sequencing; tool

Project Summary/Abstract ! Most genetic recorders described so far utilize the CRISPR/Cas9 system to leave a unique genetic scar in each cell that can be traced in daughter cells. However, since these systems are largely deletion based, there is a finite number of events and lineages that they can be used to trace. Hence all these recorders are fundamentally restricted in the number of lineages they can trace since development in mammals is a prolonged process, with radial glia in the brain dividing greater than 50 times. This central limitation precludes the utility of these approaches in mammalian model organisms that are most relevant to human development. Moreover, these technologies do not allow dynamic recording of biological events throughout the life-cycle of an animal. Thus, a different approach to lineage-tracing and biological recording is needed that does not reach saturation and will permit chronicling lineages in whole organisms and complex tissues. Here we propose to exploit recently described base-editors to enable continuous, dynamic, genetic recording in non-human primates (NHPs), such as a marmoset. Our strategy, called CHRONICLE, bypasses many of the limitations of currently used systems. CHRONICLE (Cellular Hierarchy Recording in Organisms by Nucleotide Interconversion with Cas9 Linked Editors) combines base-editing with self-targeting guide RNA arrays to generate a large repertoire of sequence variants that can be used to trace cellular hierarchy lineages in complex mammalian brains. This tool combined with single cell RNA sequencing (scRNA) will enable us to probe the developmental differences between the human and marmoset cortex and the lineage trajectory of cell types found in each species and the circuits they might contribute to. In Aim 1, we propose to use CHRONICLE to trace lineages in vitro in cortical organoids derived from human embryonic stem cells (hESCs) to trace the intrinsic properties of neural progenitors in a controlled environment. In Aim 2, we will introduce CHRONICLE into marmoset cortical organoids, derived from marmoset induced pluripotent stem cells (iPSCs), as well as marmoset pre-neurulation embryos. This will allow a comparison between in vitro and in vivo development. Finally, in Aim 3, we will use the data obtained from Aims 1 and 2 to compare developmental lineage tree in the marmoset cortex by phylogenetic reconstruction of CHRONICLE evolution in projection neurons, interneurons, and glia using scRNA analysis. This approach will permit chronicling lineages dynamically in a primate brain and bypass many of the limitations of current CRISPR-based molecular recorders. Since many stages of cortical development are unique to primates, this study has the potential to reveal novel lineage relationships and developmental milestones for the first time, and provide answers to key developmental questions. Solving the lineage tree of a primate brain will impact our ability to utilize developmental principles to restore function to diseased and damaged tissues.

项目摘要/摘要 呢 到目前为止所描述的大多数遗传记录器都利用CRISPR/CAS9系统留下独特的遗传疤痕 每个可以追溯到子细胞中的细胞。但是，由于这些系统在很大程度上是基于删除的，所以 可以使用有限数量的事件和谱系来追踪。因此，所有这些录音机都是 从根本上限制了他们可以追踪的谱系数量，因为哺乳动物的发育是 长时间的过程，大脑中的径向神经胶质大于50倍。这种中心限制排除了 这些方法与人类最相关的哺乳动物模型生物的实用性 发展。此外，这些技术不允许在整个过程中动态记录生物事件 动物的生命周期。因此，需要采取不同的谱系追踪和生物记录方法 这不会达到饱和度，并且会允许整个生物体和复杂组织中记录谱系。 在这里，我们建议利用最近描述的基础编辑以实现连续，动态，遗传记录 在非人类灵长类动物（NHP）中，例如马莫斯特。我们的策略称为编年史，绕过了许多 当前使用的系统的局限性。编年史（生物中的细胞层次结构记录 与CAS9链接编辑器的核苷酸互连）将基础编辑与自动靶向指南RNA相结合 阵列生成大量序列变体曲目，可用于跟踪细胞层次结构 复杂的哺乳动物大脑中的谱系。该工具与单细胞RNA测序（SCRNA）结合 使我们能够探究人与马尔莫斯特皮质与谱系之间的发育差异 每个物种中发现的细胞类型的轨迹以及它们可能贡献的电路。在AIM 1中，我们 建议使用编年史在源自人类胚胎的皮质器官中体外跟踪谱系 干细胞（HESC）在受控环境中追踪神经祖细胞的内在特性。目标 2，我们将编年史将纪事引入果果皮质器官，这些细节源自果胶诱导的 多能干细胞（IPSC）以及果果前延期胚胎。这将允许比较 在体外和体内发育之间。最后，在AIM 3中，我们将使用从AIMS 1和2获得的数据 通过系统发育重建，比较了摩尔果皮层中的发育谱系树 使用SCRNA分析的投影神经元，中间神经元和神经胶质的编年史演变。这种方法 将允许在灵长类动物的大脑中动态编年史谱系，并绕过许多电流的局限性 基于CRISPR的分子记录器。由于皮质发育的许多阶段是灵长类动物独有的，所以 研究有可能首次揭示新的血统关系和发展里程碑， 并为关键发展问题提供答案。解决灵长类动物大脑的谱系将影响 我们利用发育原理将功能恢复为患病和损坏的组织的能力。