PROJECT SUMMARY Dysregulated gene expression is a widespread and disease-agnostic driver of human illness. Therefore, the ability to understand and precisely control gene expression has the potential to revolutionize the therapeutic landscape. The expression of human genes is naturally controlled by an elegant convergence of regulatory forces, including the physical compaction of chromatin, post-translational modifications (PTMs) to histone proteins, DNA methylation, and the dynamic engagement between transcription factors and chromatin modifying proteins and the human genome. Although this coordinated control safeguards the balance between health and disease, our mechanistic understanding of how these regulatory forces unite to drive human gene expression, and how they can be predictably redesigned for new therapies, remains limited. Programmable epigenome editing tools based upon nuclease null CRISPR/Cas-based systems have recently emerged and enable new ways to control endogenous human gene expression and covalent modifications to native chromatin. Despite this exciting progress, major technological and conceptual gaps remain. For instance, it is mechanistically unclear how the expression levels of specific genes can be precisely tuned over wide ranges in human cells. In addition, it is incompletely understood why the same transcription factors and chromatin modifiers have different effects when localized to specific regulatory elements in different human cell types. Further, the spatiotemporal stability and functional durations of transcription factors and chromatin modifiers at different genomic loci are not well defined. The goal of this MIRA project is to overcome these conceptual and technological gaps by developing and combining diverse CRISPR/Cas-based transcription factors and chromatin modifiers with bulk and single cell epigenomics and sensitive proteomics. We will use this multidisciplinary approach to answer fundamental questions about human gene regulatory mechanisms including: (1) How can human gene expression be site- specifically controlled at the resolution observed in health and disease? (2) What are the causal functions and operational stabilities of diverse chromatin modifications? (3) To what extent does chromatin compaction, modification state, protein complex composition, and spatial proximity affect the function of transcription factors and chromatin modifiers? Altogether, our proposal has great potential to uncover and enable control over pivotal mechanisms with broad and significant importance to human health.