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

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.

Keywords: library generation; modular cloning; virology.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 3T3 Cells
  • Animals
  • Biotechnology / methods*
  • CRISPR-Associated Protein 9 / genetics
  • Cell Engineering / methods*
  • Cloning, Molecular / methods*
  • DNA / genetics
  • Ebolavirus / genetics
  • Gene Library*
  • Gene Regulatory Networks*
  • Genetic Vectors
  • HEK293 Cells
  • Humans
  • Mice
  • Plasmids / genetics
  • Synthetic Biology / methods
  • Transfection


  • DNA
  • CRISPR-Associated Protein 9