A CRISPR-based yeast two-hybrid system for investigating RNA-protein interactions

ABSTRACTDespite the great importance of RNA-protein interactions in cells, there is a very limited set of approaches available for identifying proteins that bind to a specific RNA. We report here combining the use of CRISPR technology with the yeast two-hybrid protein-protein interaction system in order to create an advantageous method for investigating RNA-protein interactions. In thisCRISPR-assistedRNA/RBPyeast (CARRY) two-hybrid system, an RNA of interest is targeted to the promoters of standard yeast two-hybrid reporter genes by fusing it to the CRISPR guide RNA in a strain expressing catalytically deactivated Cas9 (dCas9). If the promoter-tethered RNA binds to a protein fused to Gal4 transcriptional activation domain (GAD), then the reporter genes become transcribed, just as in the standard protein-protein yeast two-hybrid assay. We used the CARRY two-hybrid system to analyze MS2 bacteriophage RNA hairpin binding to the MS2 coat protein (MCP). We tested MS2 hairpin mutants with a range of biochemically determined binding affinities for MCP and found that CARRY two-hybrid detected all binding interactions with dissociation constants ≤300 nM. In summary, this new CRISPR-based yeast two-hybrid system provides an easily operable, much-needed new tool for identifying proteins that bind to a particular RNA.