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.
Animals, Base Sequence, Clustered Regularly Interspaced Short Palindromic Repeats, DNA-Binding Proteins, Deoxyribonucleases, Drosophila, Endonucleases, Genetic Engineering, HEK293 Cells, Humans, MicroRNAs, Molecular Sequence Data, Response Elements, Sequence Analysis, Transcriptional Activation, Transfection, Zebrafish