title: Understanding functional miRNA-target interactions in vivo by site-specific genome engineering.
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.
authors.affiliation: 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].
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Bassett
authors.affiliation: 1] Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK [2] [3].
authors.forename: Ghows
authors.initials: G
authors.lastname: Azzam
authors.affiliation: 1] Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK [2].
authors.forename: Lucy
authors.initials: L
authors.lastname: Wheatley
authors.affiliation: Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, UK.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Tibbit
authors.affiliation: Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Timothy
authors.initials: T
authors.lastname: Rajakumar
authors.affiliation: Computational Biology Research Group, Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Simon
authors.initials: S
authors.lastname: McGowan
authors.affiliation: Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Nathan
authors.initials: N
authors.lastname: Stanger
authors.affiliation: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm SE-106 91, Sweden.
authors.forename: Philip Andrew
authors.initials: PA
authors.lastname: Ewels
authors.affiliation: Computational Biology Research Group, Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Stephen
authors.initials: S
authors.lastname: Taylor
authors.affiliation: Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, UK.
authors.forename: Chris P
authors.initials: CP
authors.lastname: Ponting
authors.affiliation: Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, UK.
authors.forename: Ji-Long
authors.initials: JL
authors.lastname: Liu
authors.affiliation: Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Sauka-Spengler
authors.affiliation: Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Tudor A
authors.initials: TA
authors.lastname: Fulga
mesh_terms: D000818:Animals; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics* / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D003851:Deoxyribonucleases / Q000235:genetics* / Q000502:physiology; D004330:Drosophila; D004720:Endonucleases / Q000235:genetics* / Q000502:physiology; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D020218:Response Elements / Q000235:genetics* / Q000502:physiology; D017421:Sequence Analysis; D015533:Transcriptional Activation / Q000235:genetics / Q000502:physiology; D014162:Transfection; D015027:Zebrafish
chemical_list: D004268:DNA-Binding Proteins; C092744:ENA-VASP proteins; D035683:MicroRNAs; D003851:Deoxyribonucleases; D004720:Endonucleases