Bassett, A.R., Azzam, G., Wheatley, L., Tibbit, C., Rajakumar, T., McGowan, S., Stanger, N., Ewels, P.A., Taylor, S., Ponting, C.P., Liu, J.L., Sauka-Spengler, T., Fulga, T.A. (2014). Understanding functional miRNA-target interactions in vivo by site-specific genome engineering.  Nat. Commun. 5(): 4640.

FBrf0225927

Research paper

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.

PMC4143950 (PMC) (EuropePMC)