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

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 Piazza, Ni Xu, Georg A. Holländer, Thomas A. Milne, Jin-Soo Kim, Anton J. Enright, Andrew R. Bassett and Tudor A. Fulga ()
Additional contact information
Qianxin Wu: University of Oxford
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
Thomas A. Milne: University of Oxford
Jin-Soo Kim: Center for Genome Engineering, Institute for Basic Science (IBS)
Anton J. Enright: European Molecular Biology Laboratory-European Bioinformatics Institute
Andrew R. Bassett: University of Oxford
Tudor A. Fulga: University of Oxford

Nature Communications, 2017, vol. 8, issue 1, 1-17

Abstract: 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.

Date: 2017
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DOI: 10.1038/s41467-017-00686-2

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