Enabling mammalian in vivo forward genetic screens based on cell morphology

项目总结/摘要 这个项目的目标是开发一个平台，将允许细胞进行分类的基础上的属性 通过显微成像确定。虽然存在基于形态学的细胞计数器和流式细胞仪，但是这些细胞计数器和流式细胞仪不适用于任何细胞。 仪器不允许通过细胞的形态学特性来分离细胞。组合"排序 具有基于CRISPR/Cas9的诱变的“显微镜”将使体内或体外正向遗传筛选成为可能， 鉴定调节单细胞表型的遗传程序，如形状、细胞内组织， 蛋白质或细胞器亚细胞定位。 我们将进行一项概念验证研究，在该研究中，我们使用分选显微镜进行正向 基于细胞形态学的遗传筛选。心肌细胞的形式已经进化到精确地实现其功能 作用在这些结构适应中，最引人注目的可能是横小管（T-小管）， 渗透到心肌细胞中心的质膜管状内陷网络。 T-小管被认为是成熟CM的标志，并且是有效的兴奋-收缩所必需的 偶联，但很少有人知道的因素，调节T-小管的形成。使用我们的平台进行体内 基于CRISPR/Cas9的体细胞诱变和分选显微镜，我们将进行概念验证 正向遗传筛选以鉴定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来执行前向遗传 基于细胞形态的筛选。在具体目标1.A中，我们将开发一个自动化平台， 通过形态学鉴定、标记和分选细胞。这个开源硬件和软件平台 将促进广泛传播。在具体目标1.B中，我们将使用硬件/软件 该平台用于鉴定心肌细胞中T小管形成所需的基因。 为了实现这些目标，我们组建了一个跨学科的团队， （Voldman）和心脏生物学组（Pu）。沃德曼的生物工程小组在图像分析方面有专长， 自动化和基于形态学的细胞分离。Pu实验室拥有基于Cas9的体内体细胞研究的专业知识 诱变和心脏生物学。我们预计，由此产生的技术将使电力 未来遗传学将被释放在发育生物学的各种问题，与直接相关， 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.