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A New Method for Studying RNA-binding Proteins on Specific RNAs

Bio Protoc. 2021-05; 
Weiping Sun, Ziheng Zhang, Ji-Long Liu, Min Zhuang
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Recombinant Proteins … SurePAGE (GenScript, catalog number: M00657) … This work has been supported by Shanghai Municipal Science and Technology Commission (19JC1413700 to MZ) and National Natural Science Foundation of China (31922038 to MZ; 31771490 to JLL). Competing interests … Get A Quote

摘要

Proximity-based protein labeling has been developed to identify protein-nucleic acid interactions. We have reported a novel method termed CRUIS (CRISPR-based RNA-United Interacting System), which captures RNA-protein interactions in living cells by combining the RNA-binding capacity of CRISPR/Cas13 and the proximity-tagging activity of PUP-IT. Enzymatically deactivated Cas13a (dCas13a) is fused to the proximity labeling enzyme PafA. In the presence of a guide RNA, dCas13a binds specific target RNA region, while the fused PafA mediates the labeling of biotin-tagged Pup on proximal proteins. The labeled proteins can be enriched by streptavidin pull-down and identified by mass spectrometry. Here we describe the ge... More

关键词

CRISPR, CRUIS, PUP-IT, Proximity-based protein labeling, RNA-binding protein