PROJECT SUMMARY The plasticity of cell identity is evident through nuclear transfer and transcription factor (TF)-mediated reprogramming. Despite this, current reprogramming methods often yield developmentally immature and heterogeneous cell populations and therefore are unsuitable for therapeutic application or disease modeling. We seek to address the fundamental questions of why direct reprogramming is inefficient, representing a critical gap in knowledge that will be widely applicable across many cell fate engineering strategies and has broader significance for understanding how cell identity is regulated. Over the past five years of NIGMS- supported research, we have developed and applied innovative single-cell multiomic lineage tracing and novel computational technologies to dissect the mechanisms of pioneer TF-mediated reprogramming. Our proposed research builds on this work, focusing on two primary objectives: 1. Current evidence supports the hypothesis that successful reprogramming events arise from rare 'reprogramming permissive' cell types or states in which normally inaccessible target genes are engaged by ectopic TFs to drive fate change. However, the lack of understanding about the origins of reprogramming presents a significant challenge in characterizing reprogramming permissive states. We will develop and apply multiomic lineage tracing to identify the origins of successfully reprogrammed cells across various cell fate conversion strategies. Elucidating reprogramming initiation mechanisms will identify new avenues to enhance reprogramming efficiency, uncovering common and cell-type specific regulation of cell identity. 2. The identification of new, more effective reprogramming cocktails represents a current gap in cell engineering. We hypothesize that expanded cocktails of precisely delivered TFs targeting the gene regulatory networks controlling terminal cell identity will more faithfully reprogram fate. However, it is currently experimentally and computationally intractable to predict these cocktails de novo. We will use cell fusion in combination with our genomic technologies to empirically deconstruct gene regulation, providing unique insights into efficient and accurate reprogramming, informing the development of novel TF cocktails. The outcomes of this research will have significant impacts on multiple levels: a) It will facilitate improved conversion efficiency and fidelity across different cell engineering strategies, overcoming a current barrier in regenerative medicine; b) The experimental manipulation of cell fate offers a valuable model system to deconstruct and model dynamic changes in cell identity. Our study of diverse reprogramming strategies will uncover general and cell type-specific rules for cell fate specification and maintenance, providing broad biological relevance beyond cell engineering; c) We will continue to develop our innovative genomic technologies, which provide insight into the regulati...