RNA and Protein Interactomes of an RNA-Binding Protein Tagged with FLAG Epitopes Using Combinatory Approaches of Genome Engineering and Stable Transfection

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

To study the function of RNA-binding proteins (RBPs), an overexpression or knockout approach is generally used. However, as many RBPs are essential to cellular functions, the complete knockout of these proteins may be lethal to the cell. Overexpression of RBPs, on the other hand, may create an altered transcriptome and aberrant phenotypes that can mask their physiological function. Additionally, biochemical characterization of RBP often requires highly specific antibodies for efficient immunoprecipitation for downstream mass spectrometry or RNA footprinting profiling. To overcome these hurdles, we have developed a strategy to generate cellular systems either using a CRISPR-Cas9-mediated epitope tag knock-in approach or a two-step workflow to first stably express an exogenous Flag-tagged RBP and subsequently knockout the endogenous RBP using CRISPR-Cas9 gene editing. The generation of these cell lines will be beneficial for downstream RNA footprinting studies and mass spectrometry-mediated interactome studies.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages247-263
Number of pages17
DOIs
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
Volume2666
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

  • CRISPR-Cas9
  • Epitope tag knock-in
  • Formaldehyde cross-linking
  • Interactome
  • Riboproteomics
  • RNA/genetics
  • RNA-Binding Proteins/metabolism
  • CRISPR-Cas Systems/genetics
  • Transfection
  • Epitopes/genetics
  • Gene Editing/methods

PubMed: MeSH publication types

  • Journal Article

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