简介：
Overview
This protocol describes the tagging of endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. The method allows for the study of protein dynamics under endogenous regulatory control.
Key Study Components
Area of Science
- Stem Cell Biology
- Gene Editing
- Fluorescent Tagging
Background
- Endogenous tagging provides insights into protein localization and dynamics.
- Induced pluripotent stem cells (iPSCs) serve as a model for studying tagged proteins in non-transformed cells.
- The CRISPR-Cas9 system is utilized for precise genome editing.
- Fluorescent tags enable real-time observation of protein behavior.
Purpose of Study
- To generate iPSCs expressing endogenous fusion proteins with fluorescent tags.
- To enrich edited cells for further analysis.
- To explore the dynamics of proteins in various cellular contexts.
Methods Used
- CRISPR-Cas9 ribonucleoprotein complex delivery via electroporation.
- Fluorescence-activated cell sorting (FACS) for enriching edited cells.
- Clonal line generation for precise editing outcome analysis.
- Optimization of conditions for cell survival and editing efficiency.
Main Results
- Successful generation of over 35 different cell lines expressing tagged proteins.
- Knock-in efficiencies of up to 95% for targeted loci.
- Identification of monoallelic and biallelic edited clones.
- Ability to study protein dynamics in live cells.
Conclusions
- This method enhances the understanding of protein functions in live cells.
- It offers a versatile approach for studying various cellular processes.
- Potential for adaptation in other mammalian cell types.
What is the main advantage of using CRISPR/Cas9 for tagging proteins?
CRISPR/Cas9 allows for precise genome editing, enabling the introduction of fluorescent tags directly into the genome, which maintains endogenous regulation.
How are the edited cells enriched after transfection?
Edited cells are enriched using fluorescence-activated cell sorting (FACS), which isolates cells expressing the fluorescent tags.
What types of cells can this protocol be adapted for?
While this study focuses on human induced pluripotent stem cells, the protocol could be optimized for other mammalian cell types.
What is the significance of using iPSCs in this study?
iPSCs provide a diploid, non-transformed model system that can differentiate into various cell types, allowing for diverse studies of protein dynamics.
What is the expected efficiency of the knock-in process?
Knock-in efficiencies can vary, typically ranging from 0.1% to 5%, with some loci achieving up to 95% efficiency.
How does the ROCK inhibitor contribute to the protocol?
The ROCK inhibitor promotes cell survival during electroporation and FACS enrichment, enhancing the overall success of the protocol.
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