Advanced recombinase-based gene expression technology in mammalian cells

项目总结/摘要 基因表达技术与基因组工程技术是生物技术的基石 革命虽然已经取得了很多进展，但大多数基因表达控制系统被设计成 控制一个基因然而，许多生物过程，如发育和癌症进展， 是由多个基因表达的同时变化驱动的， 控制时尚随着我们的基因组编辑能力的迅速发展，基因表达的发展 控制技术已经落后。基因表达技术的进步， 因此，迫切需要时空控制来直接询问复杂的生物过程， 设计用于生物技术应用的新表型。 位点特异性DNA重组酶（SSR）（例如，Cre和Flp）已成为最强大的基因之一 哺乳动物细胞中的调控工具。我们和其他人已经证明，重组酶是唯一能够 从而产生具有高鲁棒性的异常复杂的逻辑电路。因此，重组酶代表了理想的 工程先进的基因表达控制系统的基础。大多数基于重组酶的 使用一种酶Cre设计表达技术，其提供有限的功能， 能够以时空方式独立调控的基因数量。开发下 基于重组酶的基因表达技术的产生，稳健的正交诱导型重组酶是 必要利用我们在工程诱导型重组酶和基于重组酶的 电路，我们将开发一套先进的重组酶为基础的工具，显示同时，顺序， 和/或多个所选基因表达的空间控制调谐。特别是要 目的1：开发正交小分子诱导型重组酶，用于同时控制 多个基因在同一细胞中独立表达。 目的2：开发多色光诱导基因开关，用于基因表达的空间调控 目的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。我们将开发公制 以及一个DNA储存库数据库，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，因为我们发表的专业知识，1）DNA重组酶和2） 基因电路设计这项拟议工作的成功将大大提高我们控制 在哺乳动物细胞中的基因表达，具有增强的时空精度。

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.