3D Genome Engineering: Current Advances and Therapeutic Opportunities in Human Diseases

Abstract

Dynamic chromatin 3-dimensional (3D) conformation is a key mechanism regulating gene expression and cellular function during development and disease. Elucidating the structure, functional dynamics, and spatiotemporal organization of the 3D genome requires integrating multiple experimental approaches, including chromatin conformation capture techniques, precise genome manipulation tools, and advanced imaging technologies. Notably, CRISPR/Cas systems have emerged as a revolutionary genome-editing platform, offering unprecedented opportunities for manipulating 3D genome organization and investigating disease mechanisms. This review systematically examines recent advances in CRISPR-based mammalian 3D genome engineering and explores the therapeutic potential of 3D genome engineering strategies in disease intervention.

Fig. 1.

3D genome organization hierarchy. (A) During interphase, individual chromosomes occupy discrete nuclear regions known as chromosome territories. (B) The genome divides into A (open, active, gene-dense) and B (closed, silent, gene-sparse) compartments. In the normalized contact frequency Hi-C heatmap, compartment A (active region) is displayed in pink (high-frequency interactions), while compartment B (silent region) appears in blue (low-frequency interactions). (C) TADs are chromatin subdomains with frequent internal interactions, whose boundaries are typically enriched with insulator proteins such as CTCF, as demarcated by pink rectangles. (D) Chromatin loops are formed through cohesin-mediated extrusion, which stalls upon encountering convergently oriented CBSs. This stabilizes spatial interactions between promoters and distal regulatory elements (e.g., enhancers), facilitating the recruitment of transcriptional machinery and additional regulatory factors.

Fig. 2.

Timeline of CRISPR-based imaging technologies. The field has evolved from early fluorescent labeling systems (e.g., dCas9–EGFP and dual-color fusion proteins dSpCas9-EGFP/dSaCas9-mCherry) to advanced multicolor imaging tools (e.g., CRISPRainbow and CRISPR-Sirius), significantly enhancing multicolor visualization capabilities for genomic loci. Next-generation nonrepetitive sequence imaging technologies, including dCas9-SunTag, CARGO, CRISPR-Sunspot, Casilio, CRISPR-FISHer, and CRISPRdelight, employ strategies such as signal amplification and modular protein recruitment to achieve high-resolution dynamic tracking of single-copy genomic regions. Furthermore, hybrid imaging techniques (e.g., CRISPR-LiveFISH, CAS-LiveFISH, Oligo-liveFISH, and fCRISPR) have expanded the sensitivity and applicability of imaging for investigating 3D genome dynamics in living cells.

Fig. 3.

CRISPR-based genomic imaging technologies. (A) dCas9–EGFP. Targets genomic loci via sgRNA guidance, generating sequence-specific fluorescence in live cells. (B) CRISPRainbow. Incorporates MS2/PP7/boxB RNA hairpins into sgRNAs to recruit fluorescent protein (FP) pairs, enabling multicolor imaging via spectral overlap of blue fluorescent protein (BFP)/GFP/red fluorescent protein (RFP). (C) CRISPR-Sirius. Engineered sgRNA scaffold incorporates 8×MS2 or 8×PP7 aptamers in the tetraloop region, enhancing sensitivity for repetitive sequences (≥20 copies) with 2-color labeling. (D) dCas9-SunTag. Amplifying signals via a GCN4 peptide array that recruits multiple scFv–antibody–FP conjugates enables single-copy loci imaging. (E) Casilio. Utilizes sgRNA-PBS sequences to multimerize PUF domain–FP fusions, creating localized signal hubs for nonrepetitive locus imaging. (F) CARGO. Employs a triple-plasmid system (12 sgRNAs per plasmid) for efficient and precise imaging of nonrepetitive genomic regions. (G) CRISPRdelight. CRISPR arrays encode dozens of crRNAs for parallel labeling nonrepetitive sites, achieving high-throughput live-cell imaging. (H) CRISPR-liveFISH. Integrates CRISPR targeting with FISH technology, using fluorescent sgRNA–dCas9 complexes for live-cell genomic locus visualization. (I) CAS-liveFISH. Directly image genomic loci through fluorescently labeled dCas9 proteins, providing an alternative live-cell imaging strategy. (J) CRISPR FISHer. Recruits foldon–GFP–PCP fusion proteins through sgRNA-2×PP7, inducing phase separation to form micrometer-scale condensates at target loci. (K) fCRISPR. TDeg-fused FPs are proteasomally degraded unless stabilized by binding to the pepper aptamer on target-bound sgRNAs, enabling background-free imaging.

Fig. 4.

CRISPR-based chromatin loop manipulation tools. (A) dCas9-CTCF. Artificial tethering of CTCF to a gene promoter induces chromatin loop formation and facilitates distal element-dependent transcription. (B) Heterodimer-guided looping. Engineered SpdCas9 and StdCas9 proteins fused to leucine zipper domains create programmable chromatin loops between 2 endogenous genomic loci through targeted protein dimerization, enabling precise spatial control of genomic interactions. (C) CLOuD9. ABA-inducible heterodimerization between SpdCas9-PYL1 and SadCas9-ABI creates locus-specific loops with temporal control. (D) LADL. The 470-nm light triggers dCas9-CIBN/CRY2 heterodimerization, followed by CRY2 oligomerization to stabilize loops between targeted regions. (E) BPCL. Through sgRNA engineering to incorporate specific adapter target sequences, each dCas9/sgRNA complex pair is designed with modified adapters containing matched, discrete bioorthogonal reactive groups. By employing 2 compatible bioorthogonal reaction systems (SPAAC and SPIEDAC), this platform allows for dynamic control of chromatin loop formation at multiple loci.

Fig. 5.

CRISPR-based tools for targeted chromatin tethering. (A) CRISPR-EChO. Without ABA induction, the dCas9-bound tandem repeat region adopts a flexible and open conformation. Introduction of ABA induces dCas9 heterodimerization with engineered HP1α (green). Additional engineered HP1α or endogenous HP1α (magenta) is recruited to the site via HP1α homodimerization and oligomerization. HP1α generates cis-interactions with other sites along the tandem repeat region and trans-interactions with HP1α at different loci within natural heterochromatin or additional dCas9-bound distal sites. These interactions lead to compaction of the target locus, colocalization of multiple targeted loci, and integration with natural heterochromatin. (B) CRISPR-GO. This platform combines an ABA-inducible protein dimerization module with the dCas9 targeting system to achieve reversible subnuclear compartmentalization of specific genomic loci. The system employs ABI–dCas9 fusion proteins for precise gene targeting, while PYL1 is conjugated with the inner nuclear membrane component Emerin. ABA administration induces specific ABI-PYL1 dimerization, leading to directed anchoring of target DNA sequences to the nuclear periphery. This spatial reorganization is fully reversible, with target loci returning to their original nuclear distribution upon ABA withdrawal. (C) CRISPR-PIN. The system harnesses the endogenous Coh-Doc protein interaction network. By engineering dCas9-Coh fusion proteins for locus-specific recognition and utilizing the recruitment capability of nuclear membrane-localized Esc1-Doc, this system enables precise repositioning of target DNA sequences to the nuclear periphery in yeast.

Fig. 6.

Impacts of TAD boundary aberrations on 3D genome organization and CRISPR-based correction strategies. TAD boundary abnormalities (including CTCF insertions, deletions, and TAD inversions) disrupt the spatial architecture of the 3D genome. The following CRISPR correction strategies can be used: (1) CRISPR-KO for targeted elimination of defective boundaries, (2) dCas9-CTCF fusion proteins to reprogram boundary function by artificial insulator recruitment, and (3) large-fragment insertion techniques to reconstitute chromatin loop structures. These strategies collectively enable the restoration of native TAD topology.