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

基本信息

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
项目类别：
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

项目摘要

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：光诱导的调节蛋白是询问驱动基本机制的强大工具细胞行为。为此，融合与蛋白质融合的遗传编码的光感域可能会紧密调节蛋白质活性和基因表达。虽然光诱导型分裂蛋白系统的性能很好单独的哺乳动物细胞中，很少有多种多种和正交基因调节系统存在。现存的多输入电路受到其法规类型的阻碍，并在数字上妨碍了可能的输出规模波长。我将通过创建一个红色和蓝色诱导式拆分的库来解决此限制重组酶和第一套分裂的Cas13核糖核酸酶。特定地点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互补的方法来调节可逆的模拟转录组输出。该提案中开发的多重光遗传学工具将是理解多个相互作用基因在内源信号网络中的作用的变革性。我会利用C2C12成肌细胞分化为成骨细胞或肌管的能力并进行编码依赖照明的细胞命运开关。虽然C2C12细胞一直是研究如何机械提示和生长因素的分化影响，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。