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Live-Cell Imaging of Genomic Loci Using CRISPR/Molecular Beacon Hybrid Systems

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RNA Tagging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2166))

Abstract

The ability to monitor the behavior of specific genomic loci in living cells can offer tremendous opportunities for deciphering the molecular basis driving cellular physiology and disease evolution. Toward this goal, clustered regularly interspersed short palindromic repeat (CRISPR)-based imaging systems have been developed, with tagging of either the nuclease-deactivated mutant of the CRISPR-associated protein 9 (dCas9) or the CRISPR single-guide RNA (sgRNA) with fluorescent protein (FP) molecules currently the major strategies for labeling. Recently, we have demonstrated the feasibility of tagging the sgRNA with molecular beacons, a class of small molecule dye-based, fluorogenic oligonucleotide probes, and demonstrated that the resulting system, termed CRISPR/MB, could be more sensitive and quantitative than conventional approaches employing FP reporters in detecting single telomere loci. In this chapter, we describe detailed protocols for the synthesis of CRISPR/MB, as well as its applications for imaging single telomere and centromere loci in live mammalian cells.

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Acknowledgments

This work was supported by grants from the National Key R&D Program of China (2016YFA0501603), the National Natural Science Foundation of China (Nos. 31771583 and 81371613), the Beijing Natural Science Foundation (7162114), and China’s 1000 Young Talent Award program.

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Authors and Affiliations

  1. Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, China

    Xiaotian Wu, Yachen Ying, Shiqi Mao, Christopher J. Krueger & Antony K. Chen

  2. Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA

    Christopher J. Krueger

Authors
  1. Xiaotian Wu
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  2. Yachen Ying
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  3. Shiqi Mao
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  4. Christopher J. Krueger
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  5. Antony K. Chen
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Corresponding author

Correspondence to Antony K. Chen .

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Editors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Université de Strasbourg, Strasbourg, France

    Manfred Heinlein

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Wu, X., Ying, Y., Mao, S., Krueger, C.J., Chen, A.K. (2020). Live-Cell Imaging of Genomic Loci Using CRISPR/Molecular Beacon Hybrid Systems. In: Heinlein, M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0712-1_21

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  • DOI: https://doi.org/10.1007/978-1-0716-0712-1_21

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0711-4

  • Online ISBN: 978-1-0716-0712-1

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