CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization

Abstract

SUMMARY Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30–600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.

Quantifying Three-dimensional Chromatin Organization Utilizing Scanning Transmission Electron Microscopy: ChromSTEM

“…ChromSTEM platform for analysis of chromatin organization 3D chromatin organization governs gene transcription by controlling genome connectivity, DNA accessibility, and transcriptional heterogeneity 26,27,28 . Emerging evidence shows that the proper positioning of the chromatin within the nucleus also plays an indispensable role in maintaining normal transcriptional function 10,29 . Utilizing the ChromSTEM platform, one can obtain quantitative information on the native 3D chromatin architecture.…”

“…Regulation of gene transcription is essential in sustaining normal cell function, controlling cell differentiation and determining cell fate, and transcriptional alterations have been implicated in a variety of diseases including cancer and cardiovascular, developmental, neurological, and autoimmune disorders 1,2,3 . While early studies, which focused on the molecular mechanisms of transcriptional regulation based on a linear model of genome organization, provided significant knowledge of the genome regulation, they also demonstrated the substantial limitations of this approximation 4,5,6,7,8,9,10 . Recent studies have unambiguously shown that genes can interact with multiple distal elements within distances up to several Mb away, suggesting a machinery of transcriptional regulation based on the three-dimensional (3D) chromatin structure 11,12,13,14,15 .…”

Temporal-Spatial Visualization of Endogenous Chromosome Rearrangements in Living Cells

“…Cells can be recovered after LiveFISH analysis for culture, sequencing, and functional characterization (Supplementary Figure 7). The human genome contains many repetitive sequence clusters that are highly abundant and adjacent (within 100kb) to the majority (>60%) of encoding genes, which can serve as robust chromosomal imaging regions (43). Given the targeting-flexibility of LiveFISH, our approach offers a new way to studying dynamics of translocations induced by gene editing at various endogenous genomic locations.…”

“…Given the targeting-flexibility of LiveFISH, our approach offers a new way to studying dynamics of translocations induced by gene editing at various endogenous genomic locations. The possibility of combining CRISPR LiveFISH with gene editing and other manipulation techniques (e.g., CRISPRi/a, epigenetic modification, CRISPR-GO) should further expand our capability to study the spatiotemporal dynamics of genome organization and rearrangements (16,20,(43)(44)(45)(46)(47). Figure 5D for raw sanger sequencing data.…”

CRISPR-based Live Imaging of Epigenetic Modification-Mediated Genome Reorganization

“…These methods are applied to fixed cells. On the other hand, CRISPR-based imaging provides a versatile and powerful tool to track chromatin topology in live cells in real time [10][11][12] .…”