A/Professor Tudor Fulga, WIMM - 'A multiplex genome engineering platform for in situ functional discovery of RNA cis-regulatory elements'

RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting the endogenous activity of RREs without perturbing cellular homeostasis. We have developed the genome-engineering based evaluation of RNA regulatory element activity (GenERA) approach, a CRISPR/Cas9 arrayed screening platform for high-content functional analysis of native RREs. The conceptual framework of this technology entails generation of complex sets of NHEJ-induced genomic deletions tiled over RREs, and precisely assessing the impact of each individual event on transcript abundance. We use GenERA to survey the entire regulatory landscape of a candidate 3’UTR, and apply it in a multiplex fashion to analyze combinatorial interactions between sets of miRNA response elements (MREs). We also demonstrate that GenERA is amenable to analysis of large numbers of RREs, and employ it to probe the functionality of a MRE network under cellular homeostasis.