Systematicin vivoquantification of microRNA affinities

The majority of mammalian genes are under regulation by microRNAs, yet predicting the extent of miRNA-mediated repression has remained elusive. Here we systematically quantified the biological impact of miRNAs conserved in vertebrates using stable mouse embryonic stem cell lines expressing sensitive fluorescent reporters. Differentiation of these 163 lines to the three germ layers revealed that the majority of conserved miRNAs have detectable changes in activity. We determined in vivo target affinity K D of 115 miRNAs by integrating activity measurements, CRISPR/Cas miRNA knockouts and miRNA sequencing. Target affinities of individual miRNAs spanned several orders of magnitude, with highly expressed miRNAs having overall higher K D . Scaling miRNA expression levels by their respective K D recapitulated the relative number of Argonaute-bound targets for individual miRNA families. Our results provide a rationale to determine the set of miRNAs with a biological activity in a given cell type, K D values setting expression thresholds for target repression.