Multiplexed Optogenetic Control of Mammalian Genome and Transcriptome using Recombinases and Cas13

使用重组酶和 Cas13 对哺乳动物基因组和转录组进行多重光遗传学控制

• 批准号: 10751791
• 负责人: Cristina Tous
• 金额: $ 3.92万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751791
• 关键词: Architecture; Automobile Driving; Biological; Biological Assay; Cell Fate Control; Cell Therapy; Cell physiology; Cells; Chemicals; Complex; DNA Sequence; Darkness; Degradation Pathway; Development; Diffuse; Disease model; Engineering; Enzymes; Flow Cytometry; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Geometry; Growth Factor; Guide RNA; Image Analysis; Individual; Knowledge; Libraries; Light; Lighting; Logic; Luciferases; Mammalian Cell; Memory; Modeling; Muscle Fibers; Myoblasts; Nuclear RNA; Optics; Osteoblasts; Output; Pathway interactions; Pattern; Post-Transcriptional Regulation; Proteins; RNA; RNA Degradation; RNA Interference; Regulation; Reporter; Resolution; Ribonucleases; Role; Signal Pathway; Signal Transduction; Site; Specificity; Stains; System; Transfection; Transgenes; Work; analog; cell behavior; cell growth regulation; cellular transduction; empowerment; gene interaction; gene repression; genetic regulatory protein; improved; knock-down; mammalian genome; mechanical signal; multipotent cell; nuclease; optogenetics; osteoblast differentiation; quantitative imaging; recombinase; reconstitution; screening; spatiotemporal; tissue regeneration; tool; transcription factor; transcriptome; transcriptomics; transgene expression

Abstract: Light-inducible regulatory proteins are powerful tools to interrogate the fundamental mechanisms driving cellular behavior. To this end, genetically encoded photosensory domains fused to split proteins can tightly modulate protein activity and gene expression. While light-inducible split protein systems have performed well individually, few multichromatic and orthogonal gene regulation systems exist in mammalian cells. Existing multi-input circuits are hampered by their type of regulation and the scale of possible outputs given the number of wavelengths. I will address this limitation by creating a library of red and blue light-inducible split recombinases and the first suite of split Cas13 ribonucleases. Site specific recombinases provide permanent and transgene outputs, while Cas13 ribonucleases will provide a complementary approach to modulate reversible analog transcriptomic outputs. The multiplexed optogenetic tools developed in this proposal will be transformative for understanding the role of multiple interacting genes in endogenous signaling networks. I will leverage the ability of C2C12 myoblasts to differentiate into osteoblasts or myotubes and encode an illumination-dependent cell fate switch. While C2C12 cells have been a fundamental model in studying how differentiation is impacted by mechanical cues and growth factors, this work will explore spatiotemporal control of optogenetic regulatory proteins in order to direct cell fate.

抽象的： 光诱导调节蛋白是探究驱动基本机制的强大工具 细胞行为。为此，与分裂蛋白融合的基因编码的光感应域可以紧密地 调节蛋白质活性和基因表达。虽然光诱导分裂蛋白系统表现良好 单独而言，哺乳动物细胞中存在很少的多色和正交基因调控系统。现存的 多输入电路受到其调节类型和给定数量的可能输出规模的阻碍 波长。我将通过创建红光和蓝光诱导分裂的库来解决这个限制 重组酶和第一套裂解 Cas13 核糖核酸酶。位点特异性重组酶提供永久性 和转基因输出，而 Cas13 核糖核酸酶将提供一种补充方法来调节 可逆的模拟转录组输出。本提案中开发的多重光遗传学工具将是 对于理解内源信号网络中多个相互作用基因的作用具有变革意义。我会 利用 C2C12 成肌细胞分化为成骨细胞或肌管的能力，并编码 光照依赖性细胞命运开关。虽然 C2C12 细胞一直是研究如何 分化受到机械线索和生长因子的影响，这项工作将探索时空控制 光遗传学调节蛋白以指导细胞命运。