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

Unmet Need:
DNA-sequencing technology has led to the development of many powerful techniques, such as RIP-seq and CLIP-seq, which can be used to identify many RNAs bound by a protein of interest, however, few protocols exist for identifying the proteins bound to a particular RNA. Most available techniques involve RNA pull-down followed by protein identification via mass spectrometry, which requires highly specific, robust biochemical enrichment and is prone to non-biological associations of molecules that can occur between cell lysis and affinity purification. Because so many biologically and biomedically important cellular RNA-protein interactions still remain unknown, it is advantageous to pursue their discovery using high-throughput approaches.
 
Technology Overview:
JHU researchers have developed CRISPR-assisted RNA/RBP yeast (CARRY) two-hybrid to discover proteins that bind a specific RNA. CARRY two-hybrid interrogates binding between two biological macromolecules by tethering one (the RNA) to the promoter of a reporter gene and fusing the second (the protein) to a transcriptional activation domain (see Figure). Fusion of the RNA to the CRISPR sgRNA targets the RNA to the promoters of reporter genes. Building upon the widely used yeast two-hybrid assay for protein-protein interactions, in CARRY two-hybrid, a protein fused to the transcription activation domain of Gal4 (GAD) that binds to the RNA then induces reporter gene expression, allowing selection of cell growth on culture medium lacking histidine. Thus, proteins can be selected from a GAD-fusion library — i.e., the same as existing libraries for standard two-hybrid — or direct tests can be performed between an RNA and a specific protein. The system has worked immediately on most RNA-protein interactions tested by the inventors.
 
Stage of Development:
The inventors have demonstrated that CARRY two-hybrid can readily detect RNA-protein binding interactions even with micromolar dissociation constants (K_d).
 
Publications:

• Hass, EP, and Zappulla, DC. BioRxiv, 2017

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