Light-Induced Genetic Alterations within Single Cell of a Live Vertebrate Animal

 描述：疾病状态的动物模型和用于细胞动力学原位分析的生物记者是生物医学研究的一个重要方面；然而，大多数研究集中在整个生物体或感兴趣的组织内的基因操作，这并不能准确地反映疾病状态，这些疾病状态通常与单个细胞或细胞组的缺陷有关。这种差异在很大程度上是由于在一个完整的生物体内进行单细胞操作和分析的技术挑战。虽然正在进行的SCAP研究正在表征单细胞内的转录组、蛋白质组和染色质的变化，但有必要建立一个生物系统来测试这些单细胞变化的影响。因此，与这一FOA一致，我们认为需要一种在活着的生物体的单个细胞内控制遗传操作(导致功能等位基因的丧失或外源cDNA/生物记者的表达)的系统。该系统不应改变或干扰周围环境，并应允许潜在的基于系统的分析和生物标记物的结合。因此，我们假设：1)通过使用光遗传(光诱导)Cre转基因动物，可以实现单细胞Cre重组，从而允许单细胞诱导的基因改变(即条件敲除或生物报告基因/感兴趣基因的表达)；以及2)通过使用Cre可诱导的Cas9系统，可以在完整生物体的单个细胞内快速实现单基因和多基因的去除。这两个系统将在斑马鱼胚胎内建立，但可以应用于所有模式生物。在这项提议完成后，我们将建立通用和有效的单细胞工具，用于选择性的基因改变，这将促进在许多研究领域进行单细胞分析。我们预计，这项研究将对其他一些人类疾病产生广泛的积极影响，包括(但不限于)神经生物学、免疫学、癌症生物学和发育生物学。这项建议与SCAP和FOA的目标是一致的：1)开发工具，“最大限度地减少细胞扰动，并允许细胞在一段时间内重复测量”；2)“系统级的单细胞数据集分析或建模……在组织或整个生物体的背景下；以及3)”发现新的创新工具，用于在最小扰动的情况下对生物相关的细胞群体进行时空成像、操纵、分析和建模“。

 DESCRIPTION: Animal modeling of disease states and bioreporters for in situ analysis of cellular dynamics are an essential aspect of biomedical research; however, most studies focus on genetic manipulation within the whole organism or tissue of interest, which does not accurately reflect disease states that are often associated with defects in individual cells or groups of cells. This discrepancy is largely due to the technical challenges of single cell manipulation and analysis within an intact organism. While ongoing SCAP studies are characterizing the transcriptome, proteome, and chromatin alterations within single cells, a biological system to test the impact of these single cell alterations is necessary. Therefore, consistent with this FOA, we believe that a system for con- trolled genetic manipulation (resulting in loss of function alleles or expression of exogenous cDNAs/bioreporters) within a single cell of a viable organism is required. This system should not alter or perturb the surrounding environment and should allow for potential systems based analysis and biomarker incorporation. Therefore we hypothesize that: 1) through the use of optogenetic (light inducible) Cre transgenic animals, single cell Cre recombination can be achieved allowing for single cell inducible gene alterations (i.e. conditional knockout or bioreporter/gene of interest expression); and 2) through the use of a Cre inducible Cas9 system, single and multiple gene ablations can be rapidly achieved within an individual cell of an intact organism. These two systems will be established within the zebrafish embryo but can be applied to all model organisms. Upon completion of this proposal we will have established versatile and efficient single cell tools for selective genetic alterations that will facilitate single cell analysis in a multitude of research fields. We anticipate that this research will have a broad positive impact on a number of other human diseases including (but not limited to) neurobiology, immunology, cancer biology, and developmental biology. This proposal is in alignment with the SCAP and this FOA objectives: 1) to develop tools that "minimize cell perturbation and permit viability of cells for repeated measures over time"; 2) "Systems-level single cell dataset analysis or modeling... in the context of tissues or whole organisms; and 3) "the discovery of new, innovative tools for spati- otemporal imaging, manipulation, analysis and modeling of a biologically relevant population of cells with minimal perturbation".