Enabling mammalian in vivo forward genetic screens based on cell morphology

项目摘要/摘要 这个项目的目标是开发一个平台，允许根据属性对单元格进行排序 通过显微成像确定的。虽然存在基于形态的细胞细胞仪和流式细胞仪，但这些 仪器不允许根据细胞的形态特性来分离细胞。组合“排序” 基于CRISPR/Cas9的突变将使体内或体外的遗传筛查能够 确定调节单细胞表型的遗传程序，如形状、细胞内组织和 蛋白质或细胞器的亚细胞定位。 我们将执行概念验证研究，在该研究中，我们使用分类显微镜执行转发 根据细胞形态进行遗传筛选。心肌细胞的形态已经进化到精确地履行它们的功能 角色。在这些结构适应中，最引人注目的可能是横管(T管)，即 穿透到心肌细胞中心的质膜的管状凹陷网络。 T管被认为是成熟CMS的标志，是有效兴奋收缩所必需的 偶联，但对调节T小管形成的因素知之甚少。利用我们的平台进行体内试验 基于CRISPR/Cas9的体细胞突变和分选显微镜，我们将进行概念验证 正向遗传筛查，以确定T小管形成所需的基因。 我们的具体目标是建立技术和方法学来执行正向遗传 根据细胞形态进行筛选。在具体目标1.A中，我们将开发一个自动化的平台 通过形态对细胞进行鉴定、标记和分类。这个开源的硬件和软件平台 将被设计成促进广泛传播。在具体目标1.B中，我们将使用硬件/软件 用于鉴定心肌细胞中T小管形成所需基因的平台。 为了实现这些目标，我们组建了一个由生物工程小组组成的跨学科团队。 (Voldman)和心脏生物学组(Pu)。沃尔德曼的生物工程团队在图像分析方面拥有专业知识， 自动化，以及基于形态的细胞分离。Pu实验室在基于Cas9的活体体细胞方面拥有专业知识 诱变和心脏生物学。我们预计，由此产生的技术将使 正向遗传学将在发育生物学中的各种问题上发挥作用，与 人类疾病。

Project Summary/Abstract The goal of this project is to develop a platform that will allow cells to be sorted based on properties determined by microscopic imaging. While morphology-based cell cytometers and flow cytometers exist, these instruments do not permit separation of cells by their morphological properties. Combining a “Sorting Microscope” with CRISPR/Cas9-based mutagenesis will enable in vivo or in vitro forward genetic screens to identify genetic programs that regulate single cell phenotypes such as shape, intracellular organization, and protein or organelle subcellular localization. We will perform a proof-of-concept study in which we use the Sorting Microscope to perform a forward genetic screen based on cell morphology. Cardiomyocyte form has evolved to precisely fulfill their functional role. Among these structural adaptations, perhaps the most striking are the transverse tubules (T-tubules), a network of tubular invaginations of the plasma membrane that penetrate into the center of the cardiomyocyte. T-tubules are considered a hallmark of mature CMs and are required for efficient excitation-contraction coupling, yet little is known about the factors that regulate T-tubule formation. Using our platform for in vivo CRISPR/Cas9-based somatic mutagenesis and the Sorting Microscope, we will undertake a proof-of-concept forward genetic screen to identify genes required for T-tubule formation. Our Specific Aim is to establish the technology and methodology to perform forward genetic screens based on cell morphology. In Specific Aim 1.A, we will develop a platform for automated identification, labeling, and sorting of cells by morphology. This open-source hardware and software platform will be designed to facilitate widespread dissemination. In Specific Aim 1.B, we will use the hardware/software platform to identify genes required for T-tubule formation in cardiomyocytes. To achieve these goals, we have assembled an interdisciplinary team consisting of a bioengineering group (Voldman) and a cardiac biology group (Pu). Voldman’s bioengineering group has expertise in image analysis, automation, and in morphology-based cell separation. The Pu lab has expertise in Cas9-based in vivo somatic mutagenesis and in cardiac biology. We anticipate that the resulting technology will enable the power of forward genetics to be unleashed on diverse problems in developmental biology, with direct relevance to human diseases.