Advanced recombinase-based gene expression technology in mammalian cells

项目摘要/摘要 基因表达和基因组工程技术是生物技术的基石 革命。虽然已经取得了许多进展，但大多数基因表达控制系统的设计都是为了 控制一个基因。然而，许多生物过程，如发育和癌症进展， 是由多个基因在顺序和空间上的表达同时变化所驱动的 受控制的时尚。随着我们基因组编辑能力的快速进步，基因表达的发展 控制技术已经落后了。基因表达技术的研究进展 因此，迫切需要时空控制来直接询问复杂的生物过程并 生物技术应用的工程新表型。 位点特异性DNA重组酶(SSR)(如Cre和FLP)已成为最强大的基因之一 哺乳动物细胞中的调节工具。我们和其他人已经证明了重组酶是唯一能够 创建具有高度健壮性的异常复杂的逻辑电路。因此，重组酶代表了一种理想的 先进的基因表达控制系统工程基础。大部分以重组酶为基础 表达技术是使用一种名为Cre的酶设计的，它提供的功能有限，涉及 可以以时空方式独立调节的基因数量。发展下一代 基于重组酶的基因表达技术的产生，健壮的正交可诱导重组酶 这是必要的。利用我们在工程可诱导重组酶和基于重组酶方面的丰富经验 电路，我们将开发一套先进的基于重组酶的工具，显示同时、顺序 和/或对多个选定基因的表达进行空间控制的调谐。特别是，我们将 目的1：建立可同时控制猪细小病毒感染的正交小分子可诱导重组酶 多个基因在同一细胞中独立表达。 目的2：发展用于基因表达空间调控的多通道光诱导基因开关 目标3：开发用于基因表达顺序控制的级联电路 我们将在人类和小鼠细胞中验证我们的系统，以确保广泛的适用性。我们将制定指标 和数据表，用于DNA存储库的数据库，以促进采用和共享。我的团队是独一无二的 完成这项拟议的工作，因为我们在1)DNA重组酶和2)已公布的专业知识 遗传电路设计。这项拟议工作的成功将极大地提高我们控制 增强的时空精确度在哺乳动物细胞中的基因表达。

Project Summary/Abstract Gene expression, together with genome engineering, technology are the cornerstones of the biotechnology revolution. While much advances have been made, most gene expression control systems were designed to control a single gene. However, many biological processes, such as developmental and cancer progression, are driven by the simultaneous changes of expression for multiple genes in a sequentially and spatially controlled fashion. As our genome editing capabilities rapidly progress, the development of gene expression control technology has fallen behind. Advancement in gene expression technology that enables multiplexed spatiotemporal control is therefore urgently needed to directly interrogate complex biological processes and to engineer novel phenotype for biotechnological applications. Site-specific DNA recombinase (SSR) (e.g., Cre and Flp) has become one of the most powerful gene regulation tools in mammalian cells. We and others have shown that recombinases are uniquely capable of creating exceptionally complex logic circuits with high robustness. As such, recombinase represents an ideal foundation for engineering advanced gene expression control systems. Most of the recombinase-based expression technologies were designed using one enzyme, Cre, which affords limited functionality concerning the number of genes that can be regulated independently in a spatiotemporal manner. To develop the next generation of recombinase-based gene expression technology, robust orthogonal inducible recombinases are necessary. Leveraging our vast experience in engineering inducible recombinases and recombinase-based circuit, we will develop a suite of advanced recombinase-based tools that show simultaneous, sequential, and/or spatially controlled tuning of the expression of multiple chosen genes. In particular, we will Aim 1: Develop orthogonal small molecule inducible recombinases for simultaneous control of multiple gene expressions independently in the same cell. Aim 2: Develop multichormatic light inducible gene switches for spatial control of gene expression Aim 3: Develop cascade circuits for sequential control of gene expression We will validate our system in human and mouse cells to ensure broad applicability. We will develop metric and datasheet, a database for DNA repository to facilitate adoption and sharing. My group is uniquely capable of accomplishing this proposed work because of our published expertise in 1) DNA recombinases and 2) genetic circuit designs. Success from this proposed work will dramatically increase our capability to control gene expression in mammalian cells with enhanced spatiotemporal precision.