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

摘要： 光诱导调节蛋白是询问驱动的基本机制的有力工具 细胞行为。为此，基因编码的光敏结构域融合到蛋白质中可以紧密结合在一起 调节蛋白质活性和基因表达。而光诱导分裂蛋白系统表现良好 就个体而言，哺乳动物细胞中几乎不存在多色和正交的基因调控系统。现有 多输入电路受到它们的调节类型和给定数量的可能输出的规模的限制 波长的变化。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 重组酶和第一套分裂的Cas13核糖核酸酶。位点特异性重组酶提供永久性的 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 可逆模拟转录输出。在本提案中开发的多路复用光遗传工具将是 对于了解多个相互作用的基因在内源信号网络中的作用具有变革性。这就做 利用C2C12成肌细胞分化为成骨细胞或肌管的能力并编码 依赖光照的细胞命运转换。虽然C2C12细胞一直是研究如何 分化受机械线索和生长因素的影响，这项工作将探索时空控制 光遗传调控蛋白，以指导细胞的命运。

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.