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Understanding functional miRNA–target interactions in vivo by site-specific genome engineering

Author

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  • Andrew R. Bassett

    (Medical Research Council Functional Genomics Unit, Anatomy and Genetics, University of Oxford
    Present address: Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK)

  • Ghows Azzam

    (Weatherall Institute of Molecular Medicine, University of Oxford
    Present address:School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia)

  • Lucy Wheatley

    (Weatherall Institute of Molecular Medicine, University of Oxford)

  • Charlotte Tibbit

    (Medical Research Council Functional Genomics Unit, Anatomy and Genetics, University of Oxford)

  • Timothy Rajakumar

    (Weatherall Institute of Molecular Medicine, University of Oxford)

  • Simon McGowan

    (Computational Biology Research Group, Weatherall Institute of Molecular Medicine, University of Oxford)

  • Nathan Stanger

    (Weatherall Institute of Molecular Medicine, University of Oxford)

  • Philip Andrew Ewels

    (Science for Life Laboratory, Stockholm University)

  • Stephen Taylor

    (Computational Biology Research Group, Weatherall Institute of Molecular Medicine, University of Oxford)

  • Chris P. Ponting

    (Medical Research Council Functional Genomics Unit, Anatomy and Genetics, University of Oxford)

  • Ji-Long Liu

    (Medical Research Council Functional Genomics Unit, Anatomy and Genetics, University of Oxford)

  • Tatjana Sauka-Spengler

    (Weatherall Institute of Molecular Medicine, University of Oxford)

  • Tudor A. Fulga

    (Weatherall Institute of Molecular Medicine, University of Oxford)

Abstract

MicroRNA (miRNA) target recognition is largely dictated by short ‘seed’ sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA–target interactions to the regulation of biological processes in vivo remains challenging. Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA–MRE interactions at any point during development.

Suggested Citation

  • Andrew R. Bassett & Ghows Azzam & Lucy Wheatley & Charlotte Tibbit & Timothy Rajakumar & Simon McGowan & Nathan Stanger & Philip Andrew Ewels & Stephen Taylor & Chris P. Ponting & Ji-Long Liu & Tatjan, 2014. "Understanding functional miRNA–target interactions in vivo by site-specific genome engineering," Nature Communications, Nature, vol. 5(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:5:y:2014:i:1:d:10.1038_ncomms5640
    DOI: 10.1038/ncomms5640
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