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Updated: Sep 10, 2025
Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System
Published on: May 28, 2015
Generation of two Betacellulin CRISPR-Cas9 knockout hiPSC lines to study the affected EGF system paradigm in
Agustin Cota-Coronado1, Murray Manning2, Dong-Hyung Kim1
1Department of Psychiatry, School of Clinical Sciences, Monash University, Clayton, Victoria 3168, Australia.
Abstract:
Several members of the epidermal growth factor (EGF) family have been implicated in the biology of schizophrenia (Ketharanathan et al., 2024). The EGF-related ligand, Betacellulin (BTC), plays an important role in the proliferation and differentiation of neural stem cells and our group found markedly reduced BTC levels in patients with schizophrenia. Nevertheless, the interplay of affected BTC and its participation in neural specification and neurodevelopment remains elusive. We generated Knockout (KO) - BTC clones from an existing hiPSC line through CRISPR/Cas9-mediated modification. Furthermore, we validated BTC-KO through genotyping/sequencing, FACS and Western Blot. Finally, we demonstrated trilineage differentiation potential in vitro.
Insights
Reduced Betacellulin (BTC) levels are linked to schizophrenia. This study generated BTC knockout human induced pluripotent stem cells (hiPSCs) to investigate BTC
Area of Science:
- Neuroscience
- Genetics
- Stem Cell Biology
Background:
- Epidermal Growth Factor (EGF) family members are implicated in schizophrenia.
- Betacellulin (BTC), an EGF-related ligand, is crucial for neural stem cell proliferation and differentiation.
- Reduced BTC levels were observed in schizophrenia patients, but its role in neurodevelopment is unclear.
Purpose of the Study:
- To investigate the role of Betacellulin (BTC) in neural specification and neurodevelopment.
- To generate and validate Betacellulin knockout (BTC-KO) human induced pluripotent stem cell (hiPSC) lines.
Main Methods:
- CRISPR/Cas9 gene editing was used to create BTC-KO hiPSC clones.
- Genotyping, sequencing, Flow-Activated Cell Sorting (FACS), and Western Blot were employed for validation.
- In vitro trilineage differentiation potential was assessed.
Main Results:
- Successfully generated and validated BTC-KO hiPSC clones.
- Confirmed the absence of functional BTC in the knockout lines.
- Demonstrated that BTC-KO hiPSCs retain trilineage differentiation potential.
Conclusions:
- Betacellulin knockout human induced pluripotent stem cells were successfully created and validated.
- These cells provide a model to study the function of Betacellulin in neural development and its potential link to schizophrenia.
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