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

Ishii, Takayoshi; Schubert, Veit; Khosravi, Solmaz; Dreißig, Steven; Metje-Sprink, Janina; Sprink, Thorben; Fuchs, Joerg; Meister, Armin; Houben, Andreas

doi:10.1111/nph.15720

Cited by 28 publications

(13 citation statements)

References 33 publications

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“…Among the three different protospacers used, only protospacer 1 and 2 produced signals. The same protospacer 1 was successfully used to label centromeres in fixed nuclei of A. thaliana with the help of CRISPR-FISH ( Ishii et al, 2019 ).…”

Section: Resultsmentioning

confidence: 99%

“…The same observation was made by ( Fujimoto and Matsunaga, 2017 ) for GFP-fused dCas9 imaging constructs. Intriguingly, CRISPR-imaging of centromeric and telomeric repeats works-fine on fixed nuclei and chromosomes of different plant and animal species ( Deng et al, 2015 ; Ishii et al, 2019 ; Nemeckova et al, 2019 ; Potlapalli et al, 2020 ). The in situ imaging method CRISPR-FISH (also called REGEN-ISL) is based on a fluorescence-labeled two-part guide RNA with a recombinant Cas9 endonuclease complex.…”

Section: Discussionmentioning

confidence: 99%

“…The in situ imaging method CRISPR-FISH (also called REGEN-ISL) is based on a fluorescence-labeled two-part guide RNA with a recombinant Cas9 endonuclease complex. For both imaging methods, we used telomere- and centromere-specific gRNA and A. thaliana and N. benthamiana , subsequently ( Ishii et al, 2019 ), this work). Hence, our expectation was that the selected gRNA in combination with dCas9 should also work in stably transformed plants.…”

Section: Discussionmentioning

confidence: 99%

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Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres

et al. 2020

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Development of live imaging techniques for providing information how chromatin is organized in living cells is pivotal to decipher the regulation of biological processes. Here, we demonstrate the improvement of a live imaging technique based on CRISPR/ Cas9. In this approach, the sgRNA scaffold is fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the fluorescent coat protein-tagged for MS2 and PP7 aptamers (tdMCP-FP and tdPCP-FP) are recruited to the targeted sequence. Compared to previous work with dCas9:GFP, we show that the quality of telomere labeling was improved in transiently transformed Nicotiana benthamiana using aptamer-based CRISPR-imaging constructs. Labeling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labeling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres

et al. 2020

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“…The kinetics of this reaction has been detected by the real time visualization of CRISPR/Cas9-mediated DNA labelling. The broad range of RGEN-ISL adaption to various temperatures and combinations of techniques, does have the potential for developing the chromosome biology (Ishii et al, 2019).…”

Section: Studying Structure and Functions Of Dna Sequencesmentioning

confidence: 99%

Gene Engineering of Lamiaceae Family: Basic and Applied Research

Cherednichenko¹,

Polivanova²,

Khlebnikova³

et al. 2021

revistageintec

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Gene engineering is a convenient way of solving basic and applied tasks related to the system of secondary metabolism of medicinal and aromatic plants, such as a large and economically important plant family Lamiaceae Martinov (Labiatae Juss.) that includes such major species, as Agastache, Dracocephalum, Lavandula, Mentha, Ocimum, Salvia, Satureja, Thymus, etc. The basic value of this method consists in studying the genetic mechanism of secondary metabolism, which includes key enzyme genes, transcription factors, etc. Applied research consists in producing valuable secondary metabolites with biological activity, including active pharmaceutical ingredients in both, the familyʹs plants proper and the heterologous systems using relevant plant genes. This overview considers a set of diverse gene engineering studies.

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“…The potential of Cas proteins to tolerate many different fusions without losing interaction with the sgRNA stimulated also several attempts to apply it for live imaging to localize specific sequences within cells. dCas9 protein was coupled with fluorescent tags and used to visualize genomic loci in mammals and plants (Chen et al, 2013; Nelles et al, 2016; Dreissig et al, 2017; Duan et al, 2018; Ishii et al, 2019). While so far mostly repetitive targets gave sufficient signal-to-noise ratios, first attempts with mRNA trafficking (Abudayyeh et al, 2017) are promising and suggest that this system will be adaptable in plants to study ncRNA in response to DNA damage.…”

Section: Application Of Crispr/cas Technology To Study Ncrnas In Dna mentioning

confidence: 99%

The Role of Noncoding RNAs in Double-Strand Break Repair

Durut

Scheid

2019

Front. Plant Sci.

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Genome stability is constantly threatened by DNA lesions generated by different environmental factors as well as endogenous processes. If not properly and timely repaired, damaged DNA can lead to mutations or chromosomal rearrangements, well-known reasons for genetic diseases or cancer in mammals, or growth abnormalities and/or sterility in plants. To prevent deleterious consequences of DNA damage, a sophisticated system termed DNA damage response (DDR) detects DNA lesions and initiates DNA repair processes. In addition to many well-studied canonical proteins involved in this process, noncoding RNA (ncRNA) molecules have recently been discovered as important regulators of the DDR pathway, extending the broad functional repertoire of ncRNAs to the maintenance of genome stability. These ncRNAs are mainly connected with double-strand breaks (DSBs), the most dangerous type of DNA lesions. The possibility to intentionally generate site-specific DSBs in the genome with endonucleases constitutes a powerful tool to study, in vivo, how DSBs are processed and how ncRNAs participate in this crucial event. In this review, we will summarize studies reporting the different roles of ncRNAs in DSB repair and discuss how genome editing approaches, especially CRISPR/Cas systems, can assist DNA repair studies. We will summarize knowledge concerning the functional significance of ncRNAs in DNA repair and their contribution to genome stability and integrity, with a focus on plants.

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

Cited by 28 publications

References 33 publications

Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres

Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres

Gene Engineering of Lamiaceae Family: Basic and Applied Research

The Role of Noncoding RNAs in Double-Strand Break Repair

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