CRISPR-based RNA-binding protein mapping in live cells

• DOI: 10.1016/j.bbrc.2021.10.059
• Publication Date: 2021-10-29T02:00:16Z
• Authors: Baiwen Chen; Haiyan Shi; Jia Zhang; Chun Zhou; Miaomiao Han; Wenxiu Jiang; Yuting Lai; Xia Tu; Huabin Li
• Keywords: Proteome; Guide RNA

RNA-binding proteins (RBPs) are involved in all aspects of RNA metabolism, and RNA-RBP interactions are important for cell homeostasis and viral replication. The global RNA-binding proteome was recently reported; however, little is known about the proteins that bind to specific RNAs. In this study, we describe a novel CRISPR-based RNA interaction proteomics method in live cells. In brief, dCas13a with an HA tag was expressed in cells and bound to an RNA of interest with the help of gRNA. The RNA-protein complexes physically bound to dCas13a-HA were crosslinked using UV light and captured using anti-HA beads, after which the proteins were purified and identified using mass spectrometry. We optimized this system and subsequently applied it to U1 small nuclear RNA, which revealed 226 proteins.