Advanced recombinase-based gene expression technology in mammalian cells

哺乳动物细胞中基于重组酶的先进基因表达技术

• 批准号: 10350656
• 负责人: Wilson Wong
• 金额: $ 33万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10350656
• 关键词: Adoption; Advanced Development; Basic Science; Biological Process; Biomedical Research; Biotechnology; Cells; Complex; DNA; DNA Databases; DNA Repository; Development; Engineering; Ensure; Enzymes; Foundations; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome engineering; Human; Light; Logic; Mammalian Cell; Mus; Phenotype; Preparation; Public Health; Publishing; Site; Switch Genes; System; Technology; Tissues; Work; base; design; experience; genome editing; next generation; novel; recombinase; research and development; small molecule; spatiotemporal; success; therapeutic development; tool; tumor progression

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.

项目概要/摘要 基因表达与基因组工程技术是生物技术的基石 革命。虽然已经取得了很大进展，但大多数基因表达控制系统的设计目的是 控制单个基因。然而，许多生物过程，例如发育和癌症进展， 由多个基因在顺序和空间上的表达同时变化驱动 控制时尚。随着我们的基因组编辑能力迅速进步，基因表达的发展 控制技术已经落后。基因表达技术的进步使多重分析成为可能 因此，迫切需要时空控制来直接询问复杂的生物过程并 为生物技术应用设计新颖的表型。 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节工具。我们和其他人已经证明重组酶具有独特的能力 创建具有高鲁棒性的极其复杂的逻辑电路。因此，重组酶代表了一种理想的 工程先进基因表达控制系统的基础。大多数基于重组酶 表达技术是使用一种酶 Cre 设计的，它提供的功能有限 能够以时空方式独立调控的基因数量。为开发下一个 基于重组酶的基因表达技术的产生，强大的正交诱导重组酶是 必要的。利用我们在工程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，显示同步、顺序、 和/或多个选定基因表达的空间控制调节。特别是，我们将 目标 1：开发正交小分子诱导重组酶以同时控制 多个基因在同一细胞中独立表达。 目标 2：开发多色光诱导基因开关，用于基因表达的空间控制 目标 3：开发用于顺序控制基因表达的级联电路 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。我们将开发度量 和数据表，一个 DNA 存储库数据库，以促进采用和共享。我的团队有独特的能力 完成这项提议的工作是因为我们在 1) DNA 重组酶和 2) 方面发表了专业知识 遗传电路设计。这项拟议工作的成功将极大地提高我们的控制能力 哺乳动物细胞中的基因表达具有增强的时空精度。