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Nat Commun. 2014 Aug 19;5:4640. doi: 10.1038/ncomms5640.

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

Author information

1
1] Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, UK [2] [3].
2
1] Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK [2] [3].
3
1] Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK [2].
4
Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, UK.
5
Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
6
Computational Biology Research Group, Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
7
Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm SE-106 91, Sweden.

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.

PMID:
25135198
PMCID:
PMC4143950
DOI:
10.1038/ncomms5640
[Indexed for MEDLINE]
Free PMC Article

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