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

João Pedro Fonseca; Alain R. Bonny; G. Renuka Kumar; Andrew H. Ng; Jason Town; Qiu Chang Wu; Elham Aslankoohi; Susan Y. Chen; Patrick Harrigan; Lindsey C. Osimiri; Amy L. Kistler; Hana El-Samad

One hurdle to construct genetic circuits for viral delivery is that viral vectors have a limited cargo capacity. While this issue can be bypassed by using other delivery methods, if viral delivery is the only option, smaller sized constructs that maintain function are needed. To address this need, we developed Left Connector parts (MTK Part 1) that encode ribosome skipping P2A elements to allow for the expression of 2-12 genes of interest in a multicistronic vector. To test the toolkit's ability to produce skipped proteins from one RNA, we built a multicistronic vector with one CAG promoter driving membrane-tethered iRFP713, cytoplasmic mAzamiGreen, mCerulean-tagged p38 KTR 15 , and histone H2B fused to mScarlet separated by P2A sites (Fig. 3a) . Compared to the control version of this construct, which expresses the same proteins in a multi-TU format from repeated, independent promoters, the multicistronic construct conferred a 35% reduction in vector size. Transiently transfected HEK293T cells with the multicistronic vector showed the correct localization of each of the proteins (Fig. 3b) , with no distinguishable difference from the independent multi-TU control. These data demonstrate the feasibility to deploy P2A elements to generate single multicistronic constructs that circumvent the size limitations of conventional viral delivery vectors.