DEVELOPING A CRISPR-ASSISTED RNA PURIFICATION, PROTEOMIC METHOD TO IDENTIFY RNA- SPECIFIC PROTEIN INTERACTIONS

Abstract

RNA regulation involves a complex interplay between RNA-sequence modulators and RNA-binding proteins (RBPs), many of which have yet to be identified for specific RNA species. The current methods to identify RBPs, however, are limited by their specificity, affinity and/or their ability to capture the dynamic protein interactions involved in RNA regulation. To overcome these limitations in the current RNA-RBP enrichment methods, we developed an in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)-assisted proteomic method (CARP) to enrich specific RNA and therefore, its associated RBPs. In the in vitro CARP approach, we found there was ~3-6-fold enrichment of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA), though this was accompanied by the retention of non-specific mRNA. Attempts to remove non-specifically bound species by the use of both potent and mild detergents resulted in a corresponding loss of GAPDH mRNA. In the in vivo CARP approach, we created and validated constructs expressing CRISPR components. Furthermore, we established a stable cell-line expressing halo-deactivated Cas9 and conditions for gRNA and PAMmer delivery in cells. We did not find a considerable enrichment of GAPDH mRNA with the in vivo CARP system. This is the first study to establish conditions for an in vivo CRISPR/dCas9 RNA-RBP enrichment tool and to examine, quantitatively, the RNA-specificity of the CRISPR/Cas9 system. Our findings suggest although there was enrichment of the target RNA by the in vitro CARP system, there are concerns of specificity to this approach, as dCas9 appears to promiscuously bind to RNA.