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 language | English (US) |
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Title of host publication | Methods in Molecular Biology |
Publisher | Humana Press Inc. |
Pages | 247-263 |
Number of pages | 17 |
DOIs | |
State | Published - 2023 |
Publication series
Name | Methods in Molecular Biology |
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Volume | 2666 |
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