SGRN: A Cas12a-driven Synthetic Gene Regulatory Network System

In this project, we sought to add to the available toolkit of synthetic gene regulatory network approaches by optimizing the use of dCas12a fused to a transcriptional activation domain to drive expression of genes of interest in a specified order. Conclusion: We found that nuclear localization was essential to activity of dCas12a as a transcriptional activator and that by enhancing the concentration of dCas12a-[Acitiv] in the nucleus for a previously published dCas12a activator, we could drive enhanced expression of reporter genes. Notes: Note that data transformation and gating strategy are defined in gating-ML 2.0 files provided as an attachment to this experiment. Data was generated to test a variety of optimization steps for this system. In each comparison, all conditions were tested in triplicate (with balanced batches of replicates tested as a group on different days). Therefore, a gating-ML file is provided for each replicate set (1, 2, or 3) for each comparison. All experiments included both negative ("UT", "P2-only", etc.) and positive ("PhiYFP", "mCerulean", "mCherry", etc.) controls. All experiments were conducted in triplicate to insure reproducibility.