Visualization of specific repetitive genomic sequences with fluorescent TALEs in<i>Arabidopsis thaliana</i>

Fujimoto, Shinsaku; Sugano, Shigeo S.; Kuwata, Keiko; Osakabe, Keishi; Matsunaga, Sachihiro

doi:10.1093/jxb/erw371

Cited by 35 publications

(30 citation statements)

References 56 publications

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“…Compared with an average intertelomere distance of 8.1 μm, these changes can amount to a maximum of 24.7%. Similar observations were made previously in A. thaliana by labelling telomeres via fluorescent TALEs (Fujimoto et al ., ). In contrast to Fujimoto et al .…”

Section: Resultsmentioning

confidence: 97%

“…The discovery of the Xanthomonas ‐based DNA binding domain (Boch et al ., ), which can be engineered to bind specific DNA sequences, initiated the development of transcription activator‐like effectors (TALEs) fused with fluorescence proteins (Ma et al ., ). Although fluorescently labelled TALEs were successfully used to trace genomic loci in non‐plant organisms (reviewed in Chen et al ., ), their application in plants has only recently been shown by Fujimoto et al ., ().…”

Section: Introductionmentioning

confidence: 97%

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Live‐cell CRISPR imaging in plants reveals dynamic telomere movements

et al. 2017

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SummaryElucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non‐coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long‐standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR–dCas9, we reveal dynamic telomere movements of up to 2 μm over 30 min during interphase. Furthermore, we show that CRISPR–dCas9 can be combined with fluorescence‐labelled proteins to visualize DNA–protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.

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Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 97%

Live‐cell CRISPR imaging in plants reveals dynamic telomere movements

et al. 2017

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“…The discrepancy between expected and observed signal number suggests that some telomeres are below the threshold of detectability using these approaches. In addition, a clustering of individual telomeres cannot be excluded completely, as found in A. thaliana (Schubert et al, 2012;Fujimoto et al, 2016).…”

Section: Researchmentioning

confidence: 99%

RNA‐guided endonuclease – in situ labelling (RGEN‐ISL): a fast CRISPR/Cas9‐based method to label genomic sequences in various species

et al. 2019

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Summary Visualising the spatio‐temporal organisation of the genome will improve our understanding of how chromatin structure and function are intertwined. We developed a tool to visualise defined genomic sequences in fixed nuclei and chromosomes based on a two‐part guide RNA with a recombinant Cas9 endonuclease complex. This method does not require any special construct or transformation method. In contrast to classical fluorescence in situ hybridiaztion, RGEN ‐ ISL ( RNA ‐guided endonuclease – in situ labelling) does not require DNA denaturation, and therefore permits a better structural chromatin preservation. The application of differentially labelled trans ‐activating cr RNA s allows the multiplexing of RGEN ‐ ISL . Moreover, this technique is combinable with immunohistochemistry. Real‐time visualisation of the CRISPR /Cas9‐mediated DNA labelling process revealed the kinetics of the reaction. The broad range of adaptability of RGEN ‐ ISL to different temperatures and combinations of methods has the potential to advance the field of chromosome biology.

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“…Indeed, engineered TALEs have been harnessed for a variety of applications, including genome editing (when fused to the cleavage domain of FokI nuclease or to meganucleases) 24 and design of customized transcriptional modulators 25,26 and recombinases 27 . Due to their relatively small size, fluorescently tagged TALEs have been used as small protein probes to track specific genomic DNA sequences, especially within telomeres and centromeres, in live cells 20,21,28,29 . Despite these advances, a careful validation of TALE-based imaging in different cellular systems is still needed.…”

Section: Introductionmentioning

confidence: 99%

Visualization of aging-associated chromatin alterations with an engineered TALE system

et al. 2017

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Visualization of specific genomic loci in live cells is a prerequisite for the investigation of dynamic changes in chromatin architecture during diverse biological processes, such as cellular aging. However, current precision genomic imaging methods are hampered by the lack of fluorescent probes with high specificity and signal-to-noise contrast. We find that conventional transcription activator-like effectors (TALEs) tend to form protein aggregates, thereby compromising their performance in imaging applications. Through screening, we found that fusing thioredoxin with TALEs prevented aggregate formation, unlocking the full power of TALE-based genomic imaging. Using thioredoxin-fused TALEs (TTALEs), we achieved high-quality imaging at various genomic loci and observed aging-associated (epi) genomic alterations at telomeres and centromeres in human and mouse premature aging models. Importantly, we identified attrition of ribosomal DNA repeats as a molecular marker for human aging. Our study establishes a simple and robust imaging method for precisely monitoring chromatin dynamics in vitro and in vivo.

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Visualization of specific repetitive genomic sequences with fluorescent TALEs inArabidopsis thaliana

Abstract: HighlightTranscription activator-like effectors fused to fluorescent proteins can visualize repetitive genomic sequences including centromere, telomere, and rDNA sequences for analysing chromatin dynamics in living plant cells.

Cited by 35 publications

References 56 publications

Live‐cell CRISPR imaging in plants reveals dynamic telomere movements

Live‐cell CRISPR imaging in plants reveals dynamic telomere movements

RNA‐guided endonuclease – in situ labelling (RGEN‐ISL): a fast CRISPR/Cas9‐based method to label genomic sequences in various species

Visualization of aging-associated chromatin alterations with an engineered TALE system

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