A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells
A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells
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DOI：
10.1021/acssynbio.9b00322
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发表时间：
2019-11-01
影响因子：
4.7
通讯作者：
El-Samad, Hana
中科院分区：
文献类型：
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作者：
Fonseca, Joao Pedro;Bonny, Alain R.;El-Samad, Hana
快速组装和原型细胞电路的能力对于生物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电路的方法是费力的、缓慢的和昂贵的。在这里，我们提出了哺乳动物工具包（MTK），一个基于金门的克隆工具包，用于快速，可重复和多功能的大型DNA载体组装及其在哺乳动物模型中的实现。MTK由一个精选的特征化模块化部分库组成，这些部分可以组装成转录单元，并进一步编织成复杂的电路。我们展示了MTK的能力，通过使用它来生成单整合着陆垫，创建和提供蛋白质变体和sgRNA的库，并通过基于dCas9的原型电路进行验证。作为概念的生物学证明，我们展示了MTK如何加速非感染性病毒回路的生成，从而能够快速测试对人类健康构成重大威胁的新兴病毒的药理学抑制剂。
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.