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

The ability to prototype gene circuits in synthetic biology is contingent on rapidly assessing part efficacy, optimizing and implementing changes for the next iteration. The vast combinatorics present in the MTK library present a fruitful opportunity to prototype, test, and iterate on circuit designs in parallel. To explore this idea, we constitutively expressed The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/506188 doi: bioRxiv preprint As expected, in each of the circuit iterations, expression of mAzamiGreen relative to the iRFP713 control is below basal with the repressor domain, or above basal with the activator domain (Fig. 6b) . However, the degree of activation and repression varies considerably across each circuit. More specifically, dSpCas9 achieves approximately 3-fold reduction in fluorescence through the KRAB effector and 30-fold induction in both the UAS and TRE promoters. In contrast, the dSaCas9 fused to VPR is an effective activator increasing expression 30-fold, but when fused to a KRAB domain, dSaCas9-mediated repression is comparable to background FP expression levels. The data also suggests that the TRE promoter exhibits higher basal activity due to its overall greater repression, and lower activation when compared to the UAS promoter. This information, in light of other considerations such as protein size or protospacer adjacent motif (PAM) sequence versatility, expedites the rational design and optimization of Cas9-controlled gene expression circuits through the parallelization of prototyping. As a result, this strategy can serve as a useful reference for further customized circuits, and a demonstration that this prototyping can be scaled to easily screen various combinations of genetic circuits.