Home › Cell Biology › Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
Cell Biology JoVE (Open Access) Citable · DOI

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

DOI: 10.3791/58130-v
What you'll learn
  • ✓ Transfect hiPSCs with CRISPR/Cas9 RNP complexes
  • ✓ Enrich edited cells using FACS sorting
  • ✓ Generate clonal cell lines from single cells
  • ✓ Cryopreserve clonal lines in 96-well plates
Protocol

Biopharma Insights Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

Difficulty
advanced
Total time
~3-4 weeks per cell line
Model organism
Human induced pluripotent stem cells (hiPSCs)
Biosafety
BSL-1

Steps

1
Transfect hiPSCs with Cas9 RNP

Introduce CRISPR/Cas9 ribonucleoprotein complexes into human induced pluripotent stem cells for endogenous gene tagging.

▶ 02:29
2
Enrich edited cells via FACS

Use fluorescence-activated cell sorting to enrich hiPSCs that have successfully incorporated the fluorescent tag.

▶ 05:15
3
Generate clonal hiPSC lines

Culture single putatively edited cells to establish clonal cell lines for downstream validation.

▶ 07:41
4
Cryopreserve clonal lines in 96-well plates

Freeze the established clonal hiPSC lines in a 96-well format for long-term storage and subsequent analysis.

▶ 10:36
5
Analyze results of protein tagging

Review outcomes of the endogenous tagging protocol, including efficiency and cell viability.

▶ 12:21
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