PROJECT SUMMARY The ultimate goal of this project is to create synthetic genetic circuits that accurately control the level of cell fate- specific transcription factors (TFs) autonomously in response to cell state changes. The underlying hypothesis is that the level and timing of expression of critical TFs dictates the efficiency of cell conversion protocols and the quality of produced cells. Here, we focus on the differentiation of human induced pluripotent stem cells (hiPSCs) into hemogenic endothelial cells (HECs) from which all hematopoietic stem and progenitor cells (HSC/HPCs) arise. Unfortunately, cur- rent methods to derive definite HECs (dHECs), which have the potential to produce adult-type lymphoid cells and HSCs, remain not only inefficient but are also difficult to execute and scale, and, as a consequence, exhibit high degrees of variability in outcomes between different labs, hiPSC lines, and even between replicate experiments.These problems hamper analysis of the underlying developmental processes and pose formidable obstacles to clinical translation of hiPSC-derived blood cell products since ensuring the safety and cost-effectiveness of the product necessitates high differentiation efficiency and consistency. Prior work has demonstrated that SCL (S), LMO2 (L), GATA2 (G), and ETV2 (E) TFs together are sufficient to convert hiPSCs-derived mesoderm to dHECs and that efficient forward programming requires discovery and subsequent implementation of both optimal expression levels and timing for each TF. Yet, con- ventional methods for TF-mediated cell fate programming rely on indiscriminate overexpression without any control on cellular TF levels. This is largely due to our inability to precisely control TF levels at user-defined values during cell fate programming, and this limitation has prevented discovering optimal trajectories and subsequently enforcing them. Here, we propose synthetic genetic controller circuits that overcome this hurdle. Specifically, in Aim 1, we create ge- netic circuit designs that set TF levels and use them in an efficient in vitro differentiation protocol to discover the optimal combination of S, L, G, E levels and timing. In Aim 2, we develop a new circuit architecture, based on TET1-enabled positive feedback, to prevent epigenetic silencing of our genetic circuits once we deliver them to hiPSCs. In Aim 3, we make our genetic controller circuits enforce autonomously the optimal SLGE TF levels found in Aim 1 in response to the hiPSC-to-mesoderm transition. We achieve this by a new autocatalytic ADAR-based RNA sense-and-respond system, which senses the mesoderm marker Brachyury (TBXT) and enforces user-defined TF levels in response to it. We expect that this process, by being autonomous as opposed to manual and by enforcing optimal TF trajectories, will result in a more efficient, repeatable, and robust hiPSCs to dHECs conversion protocol, thereby helping fill the gap to clinical translation. Although in this...