CRISPR based screen for small GTPase regulators of morphogenesis in Xenopus

基于 CRISPR 的非洲爪蟾形态发生小 GTP 酶调节因子的筛选

基本信息

批准号：
10354182
负责人：
Richard M Harland
金额：
$ 22.56万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-17 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10354182
关键词：
Actins Affect Antibodies Biological Assay CRISPR screen CRISPR/Cas technology Cell physiology Cells Characteristics Chimera organism Chromatin Structure Clustered Regularly Interspaced Short Palindromic Repeats Communities Complementary DNA Congenital Abnormality Craniofacial Abnormalities Cytoskeleton Data Databases Defect Development Embryo Event Failure Feedback Future GTPase-Activating Proteins Gene Expression Generations Genes Genetic Transcription Goals Guanine Nucleotide Exchange Factors Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Guide RNA Image Individual Knock-out Lead Length Light Mechanics Mediating Molecular Monomeric GTP-Binding Proteins Morphogenesis Movement Neural Tube Defects Normal tissue morphology Organism Organogenesis Phenotype Post-Transcriptional Regulation Proteins Rana Regulation Screening Result Specificity Syndrome Tertiary Protein Structure Testing Time Tissues Transcript Transcriptional Regulation Vertebrates Work XenBase Xenopus base cell behavior cell motility cell type chromatin immunoprecipitation experimental study gain of function gastrulation gene function genetic regulatory protein insight loss of function mRNA Expression mutant polymerization rho rho GTP-Binding Proteins screening tool

项目摘要

Abstract Many birth defects arise from flaws in the cell behaviors that drive normal tissue organization, including the prevalent cases of neural tube defects, and craniofacial defects. There has been considerable progress in understanding the mechanics of cell behavior; in contrast, the upstream control, at the level of cytoskeletal regulatory proteins, and the transcriptional control of expression of the regulators has been documented in fewer cases. Nonetheless, we can point to several examples where a specific morphogenetic event is ultimately regulated by the expression of genes that encode regulators of small GTPases, particularly Rho. Here we hypothesize that we will gain insights into many embryonic contexts of morphogenesis by screening directly for regulators of small GTPases that are essential for subsets of the early morphogenetic events of gastrulation and neurulation. Rather than conduct open-ended screens, we focus on GTPase Regulators, where we have argued that there is a precedent for their general importance in morphogenesis. There are about 150 Rho GAPs and GEFs listed in the community database, Xenbase, with descriptions of their temporal expression, and sometimes, their transcript localization. We will prioritize genes that are zygotically expressed to avoid regulators of general cellular processes, and then knock out targets using G0 (aka F0) Crispr, which is sufficient to identify phenotypes. Using high-throughput imaging we will screen for phenotypes associated with gastrulation and neurulation defects. Hits will then be further characterized to determine if they are locally or globally expressed. This project will ultimately generate the molecular tools to manipulate and analyze the force generation and intercellular interactions that determine how cells move in the embryo. As with the insights into neural tube defects that have come from understanding neurulation movements in frogs, we will establish principles to examine other morphogenetic events and failures that lead to birth defects.

抽象的许多出生缺陷是由驱动正常组织组织的细胞行为缺陷引起的，包括神经管缺陷和颅面缺陷的常见病例。方面已经取得了长足的进步了解细胞行为的机制；相反，上游控制，在细胞骨架水平调节蛋白以及调节因子表达的转录控制已在较少的文献中得到记录。案例。尽管如此，我们可以指出几个例子，其中特定的形态发生事件最终是受编码小 GTP 酶（尤其是 Rho）调节因子的基因表达调节。在这里我们假设我们将通过直接筛选来深入了解形态发生的许多胚胎环境小 GTP 酶的调节因子，对于原肠胚形成的早期形态发生事件的子集至关重要神经调节。我们没有进行开放式筛选，而是专注于 GTPase 调节剂，我们对此进行了争论它们在形态发生中的普遍重要性是有先例的。大约有 150 个 Rho GAP，社区数据库 Xenbase 中列出的 GEF 及其时间表达的描述，以及有时，他们的转录本地化。我们将优先考虑合子表达的基因以避免监管的一般细胞过程，然后使用 G0（又名 F0）Crispr 敲除目标，这足以识别表型。使用高通量成像，我们将筛选与原肠胚形成相关的表型和神经发育缺陷。然后，将进一步表征命中，以确定它们是本地表达还是全局表达。该项目最终将产生分子工具来操纵和分析力的产生和决定细胞在胚胎中如何移动的细胞间相互作用。就像对神经管的见解一样由于理解青蛙的神经运动而产生的缺陷，我们将建立原则检查导致出生缺陷的其他形态发生事件和失败。