CRISPR-Assisted Detection of RNA-Protein Interactions in Living Cells

Delineating the protein network associated with long non-coding RNAs (lncRNAs) is fundamental to understanding the functional mechanisms of lncRNAs. Current methods to identify lncRNA binding proteins either rely on crosslinking mediated complex co-precipitation or require extensive molecular engineering, leading to drawbacks such as loss of cellular context and low capture efficiency, and limiting their broad application. We develop a CRISPR-Assisted RNA-Protein Interaction Detection method (CARPID), which leverages CRISPR/CasRx-based RNA targeting and proximity labeling to identify binding proteins of specific lncRNA in the native cellular context. Applied to the nuclear lncRNA XIST, CARPID captured a list of known interacting proteins and multiple previously uncharacterized binding proteins. We generalize CARPID to explore binders of lncRNA DANCR and MALAT1, revealing its wide applicability in identifying RNA binding proteins. Overall design: We did not observe significant change of gene expression in cells with over expression of dCasRx-BASU and gRNAs, confirming that CARPID did not alter the physiological function of transfected cells. [GSM4285650-GSM4285655] We performed RNA-seq on immortalized mouse embryonic fibroblasts derived from a hybrid female mouse to further confirm the role of TAF15 on XCI. The hybrid mouse was a crossing of a Mus musculus (129S1/SvImJ) female and a Mus castaneus (CAST/EiJ) male. We generated RNA-seq data from this cell line in two replicates with no treatment (NT), upon 5-aza treatment, and upon 5-aza treatment in Taf15-knockdown cells [processed data: allelicExpr.readCount.matrix.csv].