Abstract

Deciphering miRNA-target Regulation by Site-specific Genome Engineering

Tudor Fulga, Associate Professor of Genome Biology, University of Oxford

microRNAs (miRNAs) provide an essential layer of gene regulation underlying cellular function, development and disease. The specificity of miRNA target recognition is largely dictated by promiscuous sequence complementarity with mRNAs (6-8nt). Consequently, computational algorithms and high-throughput biochemical approaches have predicted thousands of putative miRNA targets. Surprisingly, despite tremendous progress in understanding the complexity and importance of miRNAs, the physiological relevance of only a small proportion of miRNA-target interactions has been functionally established. Compounding the challenge, the rules governing miRNA target selection in a complex cellular environment remain poorly understood. Deciphering this fundamental aspect of miRNA biology requires novel strategies for functional interrogation of miRNA regulatory pathways in the context of a living cell. The impressive pace in the evolution of genome editing tools created an unprecedented landscape for biomedical research, and it was only a matter of time until they would impact miRNA studies. We have recently adapted CRISPR/Cas9 to functionally interrogate miRNA response elements (MREs) and assess their activity in intact biological systems. In addition, we have developed an innovative HDR strategy for assessing MRE activity in human cells. These technologies are unprecedented in their ability to reveal the contribution of MREs to miRNA-mediated post-transcriptional silencing, and can be applied to virtually all predicted MREs. Importantly, they permit the analysis of direct phenotypic consequences of blocking miRNA-MRE regulatory axes throughout development or during the adult life of an organism, which hitherto had been impractical. These powerful and versatile approaches represent a paradigm shift in the methodologies used to identify and characterize functional miRNA-target interactions in vivo.