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

To test the combinatorial assembly approach, we independently transduced the 3C cell line with 4 biological replicates of the sgRNA library. After 14 days of selection, we measured . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 tagBFP and mScarlet expression and identified cells belonging to the four possible outcomes of the viral library, suggesting that all combinatorial possibilities are achievable through this method (Fig. 5g) . Importantly, the fraction of each outcome was in accordance with the predictions of the classifier. Taken together, these data argue that the MTK is able to rapidly generate libraries of vectors that express proteins variants or multiple sgRNAs. Additionally, these libraries can be inserted into the genome by transduction or recombination and the diversity of variants, as well as the correct ratio of each variant in the library, is maintained through all cloning steps.