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.

摘要： 光诱导调节蛋白是研究光诱导蛋白的基本机制的有力工具。 细胞行为为此，与分裂蛋白融合的遗传编码感光结构域可以紧密地 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， 单独地，在哺乳动物细胞中存在很少的多色和正交基因调控系统。现有 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 的波长。我将通过创建一个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的 和转基因输出，而Cas13核糖核酸酶将提供一种互补的方法来调节 可逆模拟转录组输出。本提案中开发的多重光遗传学工具将 对于理解内源性信号网络中多个相互作用基因的作用具有变革性意义。我会 利用C2C12成肌细胞分化成成骨细胞或肌管的能力， 照明依赖性细胞命运开关。虽然C2C12细胞是研究细胞如何 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 光遗传学调节蛋白来指导细胞的命运。