3D Genome Engineering Using CRISPR/dCas Systems

The spatial organization of the genome has emerged as a central regulator of gene expression and cellular function. Chromatin architecture is organized hierarchically across multiple spatial scales and involves chromatin loops, topologically associating domains (TADs), chromatin compartments, and specialized nuclear environments that collectively shape regulatory interactions within the nucleus. Disruption of these structures contributes to a wide range of diseases, including developmental disorders, cancer, and laminopathies, stimulating growing interest in technologies capable of programmable manipulation of genome topology. The emergence of CRISPR/dCas-based technologies has transformed the field from descriptive 3D genomics to programmable genome engineering. Catalytically inactive Cas proteins fused to architectural or epigenetic effectors enable targeted manipulation of chromatin loops, loop extrusion, subnuclear positioning, and local chromatin states without altering the underlying DNA sequence. In this review, we summarize current CRISPR/dCas-based approaches for engineering three-dimensional genome architecture, discuss their mechanistic basis and applications, and highlight emerging therapeutic opportunities and major technical challenges in the field.