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Engineering 3D genome organization

Abstract

Cancers and developmental disorders are associated with alterations in the 3D genome architecture in space and time (the fourth dimension). Mammalian 3D genome organization is complex and dynamic and plays an essential role in regulating gene expression and cellular function. To study the causal relationship between genome function and its spatio-temporal organization in the nucleus, new technologies for engineering and manipulating the 3D organization of the genome have been developed. In particular, CRISPR–Cas technologies allow programmable manipulation at specific genomic loci, enabling unparalleled opportunities in this emerging field of 3D genome engineering. We review advances in mammalian 3D genome engineering with a focus on recent manipulative technologies using CRISPR–Cas and related technologies.

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Fig. 1: Hierarchical organization of the 3D genome in the nucleus.
Fig. 2: Tools for manipulating DNA looping.
Fig. 3: Tools for engineering spatial positioning of targeted genomic loci via physical tethering.
Fig. 4: Tools for engineering chromatin–nuclear body interactions.
Fig. 5: Tools for inducing interaction hubs and phase separation in the nucleus.

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Acknowledgements

L.S.Q. acknowledges support from the Pew Charitable Trusts, the Alfred P. Sloan Foundation and the Li Ka Shing Foundation. This work was partly supported by US National Institutes of Health Common Fund 4D Nucleome Program (U01 EB021240, 1U01DK127405-01).

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Glossary

Nuclear lamina

A structural filament meshwork located near the inner nuclear membrane that is composed of lamins and lamin-binding proteins that are associated with membrane proteins and chromatin.

Enhancer

A cis-regulatory DNA element that can bind to transcription factors to promote gene transcription and that may be located close to or far from its associated gene and promoter.

Promoter

A DNA element that can bind to transcription factors and RNA polymerase to initiate gene transcription. Promoters are typically located upstream of the gene that they regulate.

Chromosome conformation capture (3C)-based techniques

A set of techniques to study 3D genome organization by mapping the interaction frequency of genomic loci in three dimensions, including many technical variants such as 3C, 4C, 5C and Hi-C.

Fluorescence in situ hybridization

(FISH). An imaging technique to detect sequence-specific DNA or RNA localization in situ using fluorescently labelled oligonucleotide probes complementary to the imaged DNA or RNA.

CRISPR live-cell imaging

A technique to visualize the dynamics of specific genomic loci or RNA in living cells using CRISPR systems to enrich fluorescent components at the target DNA or RNA.

Lac operator–Lac repressor

(lacO–LacI). lacO, the operator of the bacterial Lac operon, binds tightly to the LacI protein, which represses gene transcription. Binding of isopropyl β-d-1-thiogalactopyranoside to LacI dissociates LacI from lacO.

Chromosome territory

The distinct volume of nuclear space occupied by each chromosome.

Heterochromatin

The condensed region of chromatin that is tightly packaged, which is usually gene-poor. Heterochromatin contains repressive epigenetic markers and is less accessible to transcription factors and transcription machines than is euchromatin.

Euchromatin

The less-condensed region of chromatin that is lightly packaged. Euchromatin is enriched in actively transcribed genes and more accessible to transcription factories than is heterochromatin.

Nuclear bodies

A collection of membraneless structures in the nucleus with diverse cellular functions. Different nuclear bodies are enriched in different structural proteins and RNA components.

Lamina-associated domains

The regions of chromatin that interact with nuclear lamina at the nuclear periphery. They usually contain inactive genes and can be mapped by sequencing approaches such as DamID.

Topologically associating domains

(TADs). Local self-interacting chromatin regions that are often separated by boundary elements. DNA elements exhibit higher frequency of contacts with regions within the same TAD than interactions among TADs.

Loop

A DNA loop is formed when two genome regions in the same chromosome contact, which can bring two distal DNA sites together, mediating interactions between cis-regulatory elements such as promoters, enhancers, insulators and silencers.

CTCF

(CCCTC-binding factor). A conserved 11-zinc-finger protein encoded by the CTCF gene that binds to the consensus DNA sequence CCGCGNGGNGGCAG.

Cohesin complex

A protein complex composed of four core subunits, SMC1, SMC3, RAD21 and SA1 or SA2. It can form a ring-shaped structure enclosing DNA and plays important roles in mitotic and interphase chromosome organization.

CTCF-binding sites

(CBSs). The binding sites of CCCTC-binding factor (CTCF) in the genome that contribute to the organization of topologically associating domains and loops.

Insulators

Cis-regulatory DNA elements that block inappropriate interactions between genomic regions.

Superloops

Networks of super-large loops that exist on the inactive X chromosome around the DXZ4 locus.

Optogenetic system

A light-inducible system that uses light-sensitive proteins to control downstream biological reactions in cells and in vivo.

Superenhancer

A cluster of enhancers that binds to a high level of transcription regulators and mediator complexes. Superenhancers are typically cell-type specific and can control the expression of cell-identity genes.

Liquid–liquid phase separation

(LLPS). A physical process that involves concentrated solutions of polymers (such as proteins and nucleic acids) separating into multiple aqueous phases, which can condense components into liquid droplets.

Intrinsically disordered regions

(IDRs). Proteins or protein segments that do not fold into a defined tertiary structure but can flexibly switch between different conformational states.

Low-complexity domains

(LCDs). Regions in a protein sequence that tend to form flexible and disordered structures, which may contain short amino acid repeats.

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Wang, H., Han, M. & Qi, L.S. Engineering 3D genome organization. Nat Rev Genet (2021). https://doi.org/10.1038/s41576-020-00325-5

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