Live imaging and tracking of genome regions in CRISPR/dCas9 knock-in mice

Duan, Jinzhi; Lu, Guangqing; Yu, Hong; Hu, Qingxi; Mai, Xueying; Guo, Jing; Si, Xiaofang; Wang, Fengchao; Zhang, Yu

doi:10.1186/s13059-018-1530-1

Cited by 35 publications

(27 citation statements)

References 28 publications

(26 reference statements)

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“…Additionally, nuclear morphology and chromatin organization can be altered due to experimental methodologies such as reduction in cell and nucleus volume due to fixation, change in osmolality, or as a result of culture on surfaces of various stiffness conditions [24][25][26] . Recent study that analyzed live telomere dynamics in the liver in vivo, reported significantly different dynamics from that observed in cultured cells 27,28 . It is therefore critical to reveal chromatin organization in its tissue intrinsic physiological conditions.…”

Section: Introductionmentioning

confidence: 89%

Live imaging of chromatin distribution in muscle nuclei reveals novel principles of nuclear architecture and chromatin compartmentalization

Pavlov¹,

Lorber²,

Bajpai³

et al. 2020

Preprint

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Packaging of the chromatin within the nucleus serves as an important factor in the regulation of transcriptional output. However, information on chromatin architecture on nuclear scale in fully differentiated cells, under physiological conditions and in live organisms, is largely unavailable. Here, we imaged nuclei and chromatin in muscle fibers of live, intact Drosophila larvae. In contrast to the common view that chromatin is distributed throughout the nuclear volume, we show that the entire chromatin, including active and repressed regions, forms a peripheral layer underneath the nuclear lamina, leaving a chromatin-devoid compartment at the nucleus center. Importantly, visualization of nuclear compartmentalization required imaging of un-fixed nuclei embedded within their intrinsic tissue environment, with preserved nuclear volume. Upon fixation of similar muscle nuclei, we observed an average of three-fold reduction in nuclear volume caused by dehydration and evidenced by nuclear flattening. In these conditions, the peripheral chromatin layer was not detected anymore, demonstrating the importance of preserving native biophysical tissue environment. We further show that nuclear compartmentalization is sensitive to the levels of lamin C, since over-expression of lamin C-GFP in muscle nuclei resulted in detachment of the peripheral chromatin layer from the lamina and its collapse into the nuclear center. Computer simulations of chromatin distribution recapitulated the peripheral chromatin organization observed experimentally, when binding of lamina associated domains (LADs) was incorporated with chromatin selfattractive interactions. Reducing the number of LADs led to collapse of the chromatin, similarly to our observations following lamin C over-expression. Taken together, our findings reveal a novel mode of mesoscale organization of chromatin within the nucleus in a live organism, in which the chromatin forms a peripheral layer separated from the nuclear interior.This architecture may be essential for robust transcriptional regulation in fully differentiated cells.

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Section: Introductionmentioning

confidence: 89%

Live imaging of chromatin distribution in muscle nuclei reveals novel principles of nuclear architecture and chromatin compartmentalization

Pavlov¹,

Lorber²,

Bajpai³

et al. 2020

Preprint

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“…In a later study, Gu et al extended the dCas9-EGFP approach to study the activity of non-repetitive regions in the enhancer and promoter of the FGF5 gene, each using 36 unique sgRNAs [26]. Moreover, Duan et al have demonstrated successful use of dCas9-EGFP to label highly-repetitive elements of different chromosomal loci in live mice [27]. Despite these advances, dCas9-EGFP has been observed to elicit high background signal in the nucleolus due to the tendency of the dCas9 protein to localize in the nucleolus [25], [28].…”

Section: Crispr/deactivated Crispr-associated Protein 9 a Powerful Tmentioning

confidence: 99%

Progress and Challenges for Live-Cell Imaging of Genomic Loci using CRISPR-Based Platforms

Mao

Ying

et al. 2019

Genomics, Proteomics &Amp; Bioinformatics

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Chromatin conformation, localization, and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states, the requirement for cell fixation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context. In this review, we describe currently-available approaches for imaging single genomic loci in cells, with special focus on clustered regularly interspaced short palindromic repeats ( CRISPR )-based imaging approaches. In addition, we discuss some of the challenges that limit the application of CRISPR-based genomic imaging approaches, and potential solutions to address these challenges. We anticipate that, with continued refinement of CRISPR-based imaging techniques, significant understanding can be gained to help decipher chromatin activities and their relevance to cellular physiology and pathogenesis.

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“…A number of groups have used dCas9 imaging systems to track the dynamics of specific genes, regulatory elements (e.g., telomeres, centromeres, enhancers and promoters), or individual chromosomes (Knight et al, 2015 ; Zhou et al, 2017 ; Gu et al, 2018 ). In addition to mammalian cells, CRISPR-based imaging tools have also been applied to label DNA in other species, including yeast, plant and mouse cells (Dreissig et al, 2017 ; Duan et al, 2018 ; Xue and Acar, 2018 ; Han et al, 2019 ). Besides live-cell DNA tracking, dCas systems, including dCas9 and dCas13, have been engineered to monitor RNA dynamics.…”

Section: Development Of Dcas Platform For Imagingmentioning

confidence: 99%

Recent advances in CRISPR research

et al. 2020

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The clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized life sciences and developed rapidly. Here, we highlight the recent advances in development and application of CRISPR technologies, including the discovery of novel CRISPR systems, CRISPR base editing and imaging, and the applications of CRISPR in plant breeding, animal breeding, disease modeling and biotherapy.

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Live imaging and tracking of genome regions in CRISPR/dCas9 knock-in mice

Cited by 35 publications

References 28 publications

Live imaging of chromatin distribution in muscle nuclei reveals novel principles of nuclear architecture and chromatin compartmentalization

Live imaging of chromatin distribution in muscle nuclei reveals novel principles of nuclear architecture and chromatin compartmentalization

Progress and Challenges for Live-Cell Imaging of Genomic Loci using CRISPR-Based Platforms

Recent advances in CRISPR research

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