CAS-LiveFISH: Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos

Geng, Yongtao; Pertsinidis, Alexandros

doi:10.1101/2020.08.25.265306

Cited by 1 publication

(4 citation statements)

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“…The next decade will likely see a continued expansion of imaging-driven techniques with a strong emphasis on multiplexing and on live microscopy, especially in conjunction with sub-diffractive resolution. As already now we see the implementation of Live FISH, further development and specialization of these methods could possibly help to study enhancer-promoter dynamics in respect to transcriptional output 206,207 . Furthermore, live-microscopy-based techniques could be well suited to study the kinetics of transcription factor binding to chromatin, a subject that is poorly understood.…”

Section: Future Directionsmentioning

confidence: 92%

“…Second, the signal must be sufficiently strong in order to visualize individual loci. Multiple different methods have been developed to reach this goal 49,50,[204][205][206][207] . Chimeric array of gRNA-oligo (CARGO) and CRISPR-Cas-mediated…”

Section: Emerging Genome Structure Technologiesmentioning

confidence: 99%

“…Furthermore, the use of catalytically active Cas9 together with dCas9 allowed the simultaneous visualization of DSBs and fluorescent DSB-repair proteins, practically creating a live Immuno-FISH 206 . Finally, Live FISH was further expanded by coupling the dCas9-gRNAs with the dCas13-gRNA system, thereby granting visualization of both DNA and nascent RNA transcripts in live cells 206,207 . Together, these and similar dCas9-based techniques might become valuable for studying chromatin and transcription dynamics in live cells, and open venues for application ranging from basic science to diagnostics 50,208 .…”

Section: Emerging Genome Structure Technologiesmentioning

confidence: 99%

“…Multiple different methods allow the study of chromatin in 4D (changes of 3D chromatin structure over time) 50,[204][205][206][207] . Here, we illustrate two such methods based on nuclease-dead Cas9…”

Section: Figurementioning

confidence: 99%

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Understanding 3D genome organization by multidisciplinary methods

Jerković

Cavalli

2021

Nat Rev Mol Cell Biol

262

172

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Understanding how chromatin is folded in the nucleus is fundamental to understanding its function. Although 3D genome organization has been historically difficult to study owing to lack of relevant methodologies, major technological breakthroughs in genome-wide mapping of chromatin contacts and advances in imaging technologies in the 21 st century considerably improved our understanding of chromosome conformation and nuclear architecture.In this Review, we discuss methods of 3D genome organization analysis, including sequencing-based techniques, such as Hi-C and its derivatives, micro-C, DamID and others; microscopy-based techniques, such as super-resolution imaging coupled with fluorescent in situ hybridization (FISH), multiplex FISH, in situ genome sequencing and live microscopy methods; and computational and modeling approaches. We describe the most commonly used techniques and their contribution to our current knowledge of nuclear architecture and, finally, we provide a perspective on up-and-coming methods that open possibilities for future major discoveries.Simultaneously with the development of the C-based techniques, ligation-independent techniques were invented to assay not only chromosome conformation in general, but also the nuclear position of chromatin contacts (tyramide signal amplification (TSA), DNA adenine methyltransferase identification (DamID), split-pool recognition of interactions by tag extension (SPRITE)) and multi-way contacts (SPRITE and genome architecture mapping (GAM)), which are not assayed effectively using ligation-based techniques [34][35][36][37][38][39][40][41][42] . Finally, the recent advancement of superresolution microscopy and imaging techniques allowed us to investigate chromatin conformation of single cells at extremely high resolution and at a higher throughput than ever before 12,14,15,[43][44][45][46][47][48] . In addition to improvements in spatial resolution, live-imaging in combination with genome-engineering using CRISPR-Cas9 systems facilitated and improved the study of chromatin-contact dynamics [49][50][51] .Owing to these methodological and technological advancements, it is not surprising that the past decade has provided major revelations in 3D genome organization and function. Most notably is the finding that chromosomes in interphase predominantly fold into two compartments, A and B, which respectively consist of predominantly gene-active and gene-inactive regions 27 (Figure 1). Furthermore, parts of compartments, from the same or different chromosomes, can come together and create hubs, which are connected by multiple chromatin interactions, thereby sharing a common function (for example, gene repression) and coalescing around different nuclear bodies such as nuclear speckles 35,37,52 . On a scale below the compartments, chromatin interactions were found to be enriched within domains of 100 kb to 1 Mb in length termed topologically associating domains (TADs); these partially insulated domains are subdivided into smaller chromatin nanodomains (CNDs) 43,53-57 ...

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Section: Future Directionsmentioning

confidence: 92%