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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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Efficient bi-allelic tagging in human induced pluripotent stem cells using CRISPR.
Xingjie Ren1, Maya Asami Takagi1, Yin Shen2
1Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA.
STAR Protocols
|February 28, 2023
Summary
We developed an efficient CRISPR-Cas9 method for bi-allelic tagging of genes in human induced pluripotent stem cells (iPSCs). This technique allows studying gene function in its natural genomic environment.
Area of Science:
- Molecular Biology
- Genetics
- Stem Cell Biology
Background:
- Studying endogenous gene function requires methods that preserve the native genomic context.
- CRISPR-Cas9 technology offers precise genome editing capabilities.
Purpose of the Study:
- To present an efficient protocol for bi-allelic tagging of protein-coding genes.
- To enable the study of gene function and transcriptional control in human induced pluripotent stem cells (iPSCs).
Main Methods:
- CRISPR-Cas9-mediated homology-directed repair for gene tagging.
- Design and cloning of fluorescent reporter constructs.
- Electroporation and single-cell clone isolation and validation in iPSCs.
Main Results:
- An efficient protocol for bi-allelic tagging of endogenous genes was established.
- The method successfully integrated fluorescent reporters into protein-coding genes in human iPSCs.
Conclusions:
- The described tagging strategy is effective for studying gene function in the native genomic context.
- This protocol is adaptable for knockin of various reporters in diverse cell types for biomedical research.
