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

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, Tatjana Sauka-Spengler and Tudor A. Fulga ()
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Andrew R. Bassett: Medical Research Council Functional Genomics Unit, Anatomy and Genetics, University of Oxford
Ghows Azzam: Weatherall Institute of Molecular Medicine, University of Oxford
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

Nature Communications, 2014, vol. 5, issue 1, 1-11

Abstract: 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.

Date: 2014
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DOI: 10.1038/ncomms5640

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