A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

• DOI: 10.1021/acssynbio.9b00322
• Publication Date: 2019-11-05T15:39:10Z
• Authors: João Pedro Fonseca; Alain R. Bonny; G. Renuka Kumar; Andrew H. Ng; Jason Town; Qiu Chang Wu; Elham Aslankoohi; Susan Y. Chen; Galen Dods; Patrick Harrigan; Lindsey C. Osimiri; Amy L. Kistler; Hana El-Samad
• Keywords: 0303 health sciences; Genetic Vectors; 3T3 Cells; DNA; Ebolavirus; Transfection; 3. Good health; Mice; 03 medical and health sciences; HEK293 Cells; CRISPR-Associated Protein 9; Animals; Humans; Gene Regulatory Networks; Synthetic Biology; Cloning, Molecular; Cell Engineering; Biotechnology; Gene Library; Plasmids

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.