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Updated: Jun 12, 2025
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Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
Published on: August 25, 2018
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Protocol for CRISPR-based endogenous protein tagging in mammalian cells
Yu-Xi Xiao1, Jiarun Wei1, Jason Moffat2
1Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
STAR Protocols
|October 20, 2024
Summary
This study introduces a CRISPR-based method for endogenous protein tagging in mammalian cells, enabling researchers to track protein localization and interactions. The protocol details tagging specific human proteins, offering a flexible approach for functional studies.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Understanding protein function requires tracking protein localization and interactions within cells.
- Endogenous protein tagging is a powerful technique for studying native protein behavior.
Purpose of the Study:
- To present a detailed protocol for CRISPR-based endogenous protein tagging in mammalian cells.
- To provide a flexible and broadly applicable method for functional proteomic studies.
Main Methods:
- CRISPR-Cas9 and Cas12a guide design for gene knockin.
- Modular repair template design and cloning.
- Lipid transfection and electroporation for delivering CRISPR components.
- Adaptability for both plasmid and ribonucleoprotein complex (RNP) formats.
Main Results:
- Successful endogenous tagging of human TSC22D2 and MAP4 proteins.
- Demonstration of a versatile CRISPR-based endo-tagging protocol.
- The protocol accommodates various Cas nuclease delivery methods.
Conclusions:
- The developed CRISPR-based endo-tagging protocol offers flexibility and broad applicability for studying endogenous proteins.
- This method facilitates the investigation of protein localization and proximal interactions.
- The protocol serves as a valuable resource for researchers in molecular and cell biology.
