CRISPR-based RNA-binding protein mapping in live cells

Baiwen Chen; Haiyan Shi; Jia Zhang; Chun Zhou; Miaomiao Han; Wenxiu Jiang; Yuting Lai; Xia Tu; Huabin Li

doi:10.1016/j.bbrc.2021.10.059

CRISPR-based RNA-binding protein mapping in live cells

Biochem Biophys Res Commun. 2021 Oct 29:583:79-85. doi: 10.1016/j.bbrc.2021.10.059. Online ahead of print.

Authors

Baiwen Chen¹, Haiyan Shi², Jia Zhang¹, Chun Zhou¹, Miaomiao Han¹, Wenxiu Jiang¹, Yuting Lai¹, Xia Tu³, Huabin Li⁴

Affiliations

¹ ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital,Fudan University, Shanghai, 200031, China.
² Department of ER, CHINA RESOURCES & WISCO GENERAL HOSPITAL, Wuhan University of Science and Technology, No.209 Metallurgical Avenue, Qingshan District, Wuhan, Hubei, 430080, China.
³ Department of ER, CHINA RESOURCES & WISCO GENERAL HOSPITAL, Wuhan University of Science and Technology, No.209 Metallurgical Avenue, Qingshan District, Wuhan, Hubei, 430080, China. Electronic address: hrwg_tx@crmedical.hk.
⁴ ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital,Fudan University, Shanghai, 200031, China. Electronic address: huabin.li@fdeent.org.

PMID: 34735883
DOI: 10.1016/j.bbrc.2021.10.059

Abstract

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.

Keywords: RNA-Binding protein; U1 snRNA; dCas13a.