Every cell within our body carries the same genetic information (DNA), yet following iterative developmental transitions hundreds of morphologically and functionally distinct cell types are being generated. At the foundation of this fascinating cellular diversification lies a milieu of finely orchestrated and sophisticated regulatory programmes, which act to turn on or off thousands of genes (~20,000 in humans) with minute spatial and temporal precision. Errors in these programmes can give rise to developmental defects and many human diseases including cancer. One such essential regulatory layer is provided by microRNAs (miRNAs). miRNAs are fascinating RNA molecules comprised of only 21 to 23 letters of the genetic alphabet, and are never translated into a polypeptide chain (protein). Strikingly, these tiny pieces of RNA appear to act as molecular rheostats of gene expression programs (protein production) in almost every living organism, by binding to numerous messenger RNAs (mRNAs) via defined "miRNA response elements" (MREs). Therefore, central to understanding cellular pathways regulated by miRNAs, is identification of their direct functional targets (MREs) in vivo. Paradoxically, this essential facet of miRNA biology has met with relatively limited success to date. Due to the complexity of the cellular environment, identifying all functional targets of a miRNA in the context of a living cell requires a systems biology approach, which hitherto had been technically unfeasible. Here, we propose to develop an innovative high-throughput discovery pipeline, which will enable for the first time a systems-level functional characterization of any primary miRNA target network. At the core of this revolutionary approach lies the development and assembly of two cutting edge technology platforms: i) a multiplex genome engineering-based strategy for assessing MRE activity; ii) a platform for parallel interrogation of miRNA target site accessibility. Integration of these tools in a combinatorial mode will engender unprecedented insight into the principles and rules governing miRNA target selection in vivo, thus addressing this fundamental, yet unmet, dimension in miRNA biology. Therefore, we anticipate that this endeavour will have a major impact on the research community, empowering scientists with the ability to understand, predict, and assess the impact of miRNAs in the context of a living cell. Considering the interdisciplinary nature underlying this research, the proposal brings together four leading international research centres: Weatherall Institute of Molecular Medicine Oxford, Genome Engineering Centre Oxford, Oxford Genomics Centre (WTCHG), and the EMBL European Bioinformatics Institute (EMBL-EBI) Cambridge.