Project Summary Pancreatic beta cells derived from human induced pluripotent stem cells (hiPSCs) hold the potential to dramatically change the treatment of type 1 diabetes (T1D). To realize the therapeutic application of these cells, however, we need to develop a robust and rapid means for generating beta cells. Here, the investigators propose a novel paradigm for direct programming of hiPSC into beta cells via CRISPR activation of transcription factors (TFs). These CRISPR activation effectors will be delivered into hiPSCs using modRNAs, avoiding genomic integrations by previous viral vector approach. Furthermore, the investigators will also use recent advances in gene editing to generate universal donor hiPSC clones, which will be used for beta cell programming. These universal donor beta cells can be used for allogeneic transplantation without immune rejections. First, the investigators will investigate optimal msgRNAs for activation of pancreatic TFs in hiPSCs. Next, they will use a multiplexed msgRNA expression system to express all msgRNAs to activate numerous msgRNAs coupled with the dCas9 activator modRNA to study beta cell programming. In Aim 3, the investigators will generate immune-privileged beta cells via programming of universal donor hiPSCs. Universal donor hiPSCs will be generated by removing B2M to inhibit T cell-mediated rejection, and by overexpression of HLA-E trimer to avoid the natural killer (NK) cell-mediated lysis of transplanted cells. The investigators will then use in vitro and in vivo assays to characterize programmed beta cells. For in vitro experiments, the investigator will perform glucose stimulated insulin secretion assay. For in vivo assays, the investigator will use the NRG Akita mice to test whether programmed beta cells can reverse diabetes in vivo. The investigator will also use humanized NSG mice to study whether programmed beta cells from universal donor hiPSCs exhibit immune evasive properties as compared to cells generated from normal hiPSCs.