Fonseca João Pedro, Bonny Alain R, Kumar G Renuka, Ng Andrew H, Town Jason, Wu Qiu Chang, Aslankoohi Elham, Chen Susan Y, Dods Galen, Harrigan Patrick, Osimiri Lindsey C, Kistler Amy L, El-Samad Hana
Department of Biochemistry and Biophysics, California Institute for Quantitative Biosciences , University of California, San Francisco , San Francisco , California 94158 , United States.
Chan Zuckerberg Biohub , San Francisco , California 94158 , United States.
ACS Synth Biol. 2019 Nov 15;8(11):2593-2606. doi: 10.1021/acssynbio.9b00322. Epub 2019 Nov 5.
The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods for the assembly of mammalian DNA circuits are laborious, slow, and expensive. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible, and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be assembled into transcriptional units and further weaved into complex circuits. We showcase the capabilities of the MTK by using it to generate single-integration landing pads, create and deliver libraries of protein variants and sgRNAs, and iterate through dCas9-based prototype circuits. As a biological proof of concept, we demonstrate how the MTK can speed the generation of noninfectious viral circuits to enable rapid testing of pharmacological inhibitors of emerging viruses that pose a major threat to human health.
快速组装细胞电路并制作其原型的能力对于生物学研究及其在生物技术和医学中的应用至关重要。目前用于组装哺乳动物DNA电路的方法既费力、耗时又昂贵。在此，我们展示了哺乳动物工具包（MTK），这是一种基于金门克隆的工具包，用于快速、可重复且通用地组装大型DNA载体，并将其应用于哺乳动物模型。MTK由一个经过精心策划的、具有特征的模块化元件库组成，这些元件可以组装成转录单元，并进一步编织成复杂的电路。我们通过使用MTK来生成单整合着陆平台、创建和递送蛋白质变体库和sgRNA库，以及迭代基于dCas9的原型电路，展示了MTK的功能。作为概念的生物学验证，我们展示了MTK如何加速非感染性病毒电路的生成，以便能够快速测试对人类健康构成重大威胁的新型病毒的药理抑制剂。
