High-throughput single-transcription factor-mediated direct cell-fate engineering

Abstract Engineering cell identity has transitioned from descriptive developmental biology into a predictable, quantitative discipline. Central to this transition is the discovery that single transcription factors (TFs) can act as master regulators, capable of bypassing hierarchical developmental constraints to directly reprogram cell fate. Although early studies relied on candidate-based approaches, recent technological advances in high-throughput methods and single-cell multi-omics have revolutionized the field. In this Review, we focus on the emergence of large-scale single-TF overexpression screens as a discovery engine for lineage regulators. We discuss how systematic overexpression screens, particularly in pluripotent stem cell systems, have enabled the identification of single TFs with cell-fate reprogramming potential. We examine the shift from bulk transcriptomics to single-cell trajectory inference, which reveals the inherent heterogeneity, rapid kinetics, and stochastic nature of fate reprogramming. Furthermore, we interrogate the mechanistic features that need to be considered during fate transitions, such as pioneer factor activity, chromatin competence, and dosage-dependent lineage bifurcations. By integrating large-scale TF overexpression data with multi-omic readouts, we discuss principles for cell-fate engineering and define successful conversion through transcriptional stability, epigenetic memory, and functional maturity. This systematic perspective provides a roadmap for the precise generation of therapeutic cell types in regenerative medicine.