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, Tudor A Fulga

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)4640
JournalNature Communications
Volume5
DOIs
Publication statusPublished - 19 Aug 2014

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