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

项目概要/摘要 ! 迄今为止描述的大多数遗传记录器都利用CRISPR/Cas9系统在基因组中留下独特的遗传疤痕。 每个细胞都可以在子细胞中追踪。然而，由于这些系统主要基于删除， 它们可以用来追踪的事件和世系是有限的。因此，所有这些记录器都是 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一个 延长过程，大脑中放射状胶质细胞分裂超过50次。这一核心限制排除了 这些方法在与人类最相关的哺乳动物模式生物中的效用 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，需要一种不同的方法来追踪血统和生物记录 它不会达到饱和，并允许在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器，使连续的，动态的，遗传记录 在非人灵长类动物（NHP）中，例如绒猴。我们的战略，称为CHRONICLE，绕过许多 目前使用的系统的局限性。CHRONICLE（生物体细胞层次记录， 与Cas9连接的编辑器的核苷酸互换）将碱基编辑与自靶向引导RNA组合 阵列，以生成可用于追踪细胞层次结构的大量序列变体 复杂哺乳动物大脑中的谱系。该工具与单细胞RNA测序（scRNA）相结合， 使我们能够探索人类和绒猴皮层之间的发育差异， 在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。目标1： 我建议使用CHRONICLE在体外追踪人胚胎皮质类器官中的谱系 干细胞（hESC）在受控环境中追踪神经祖细胞的内在特性。在Aim中 2、我们将CHRONICLE导入绒猴皮层类器官，衍生自绒猴诱导的 多能干细胞（iPSC）以及绒猴神经形成前胚胎。这将允许比较 在体外和体内发育之间。最后，在目标3中，我们将使用从目标1和2中获得的数据 通过系统发育重建比较绒猴皮层的发育谱系树， 使用scRNA分析的投射神经元、中间神经元和神经胶质的慢性进化。这种方法 将允许在灵长类动物大脑中动态地记录谱系，并绕过电流的许多限制。 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所独有的， 这项研究有可能首次揭示新的谱系关系和发育里程碑， 并提供关键发展问题的答案。解决灵长类大脑的谱系树将影响 我们利用发育原理恢复患病和受损组织功能的能力。

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.