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In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering

Author

Listed:
  • Qianxin Wu

    (University of Oxford
    Wellcome Trust Sanger Institute)

  • Quentin R. V. Ferry

    (University of Oxford)

  • Toni A. Baeumler

    (University of Oxford)

  • Yale S. Michaels

    (University of Oxford)

  • Dimitrios M. Vitsios

    (European Molecular Biology Laboratory-European Bioinformatics Institute)

  • Omer Habib

    (Center for Genome Engineering, Institute for Basic Science (IBS))

  • Roland Arnold

    (University of Oxford)

  • Xiaowei Jiang

    (University of Oxford)

  • Stefano Maio

    (University of Oxford)

  • Bruno R. Steinkraus

    (University of Oxford)

  • Marta Tapia

    (University of Oxford)

  • Paolo Piazza

    (Wellcome Trust Centre for Human Genetics)

  • Ni Xu

    (University of Oxford)

  • Georg A. Holländer

    (University of Oxford
    University of Basel)

  • Thomas A. Milne

    (University of Oxford)

  • Jin-Soo Kim

    (Center for Genome Engineering, Institute for Basic Science (IBS)
    Seoul National University)

  • Anton J. Enright

    (European Molecular Biology Laboratory-European Bioinformatics Institute)

  • Andrew R. Bassett

    (University of Oxford
    Wellcome Trust Sanger Institute)

  • Tudor A. Fulga

    (University of Oxford)

Abstract

RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3′UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment.

Suggested Citation

  • Qianxin Wu & Quentin R. V. Ferry & Toni A. Baeumler & Yale S. Michaels & Dimitrios M. Vitsios & Omer Habib & Roland Arnold & Xiaowei Jiang & Stefano Maio & Bruno R. Steinkraus & Marta Tapia & Paolo Pi, 2017. "In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering," Nature Communications, Nature, vol. 8(1), pages 1-17, December.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_s41467-017-00686-2
    DOI: 10.1038/s41467-017-00686-2
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