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Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
Published on: August 25, 2018
Establishment of TUBB3-mCherry knock-in human pluripotent stem cell line using CRISPR/Cas9 (SNUe003-A-4)
A-Hyeon Kim1, Ha Myoung Lee1, Hong-Sik Kim1
1Department of Biomedical Sciences, College of Medical Convergence, Catholic Kwandong University, Gangneung-si, Gangwon-do, Republic of Korea.
Abstract:
TUBB3 is a structural neuronal protein important for multiple neuronal functions including axonal guidance and maturation. This study aimed to generate a human pluripotent stem cell (hPSC) line with a TUBB3-mCherry reporter using CRISPR/SpCas9 nuclease. The stop codon in the last exon of TUBB3 was replaced with a T2A-mCherry cassette using CRISPR/SpCas9-mediated homologous recombination. The established TUBB3-mCherry knock-in cell line exhibited typical pluripotent characteristics. The mCherry reporter faithfully replicated the endogenous level of TUBB3 upon induction of neuronal differentiation. The reporter cell line could contribute to the investigation of neuronal differentiation, neuronal toxicity, and neuronal tracing.
