CRISPR/Cas9-Based Modeling of JAK2 V617F Mutation in K562 Cells Reveals Enhanced Proliferation and Sensitivity to Therapeutic Agents
Abstract
:1. Introduction
2. Results
2.1. Generation and Characterization of K562 Cell Lines Expressing Wild Type and Mutated JAK2 Cells
2.2. JAK2 Gene Expression and Cell Proliferation
2.3. Effects of IFN-α2a and Arsenic Trioxide on Cells with the Mutated JAK2 V617F Gene
2.4. Effect of Phorbol 12 Myristate 13 AcetateInduced Differentiation of Modified Cells to Megakaryocytes
3. Discussion
4. Materials and Methods
4.1. Establishment of the K562 Cell Line with CRISPR/Cas9 Expression of the JAK2V617F Mutation
4.2. Characterization of Genetically Modified K562 Cell Lines
4.3. RT-qPCR Analysis
4.4. Effects of Drugs on Cell Lines in the Presence of the JAK2 V617F Mutation
4.5. PMA-Induced Megakaryocyte Differentiation
4.6. Statistical Analyses
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
JAK2 | Janus kinase 2 |
MPNs | Myeloproliferative neoplasms |
IFN-α2a | Interferon α2a |
PV | Polycythemia vera |
ET | Essential thrombocythemia |
PMF | Primary myelofibrosis |
TPO | Thrombopoietin |
EPO | Erythropoietin |
Cas9 | CRISPR-associated protein 9 |
DSBs | Double-strand breaks |
RNP | Ribonucleoprotein |
IC50 | The half-maximal inhibitory concentration |
ROS | Reactive oxygen species |
DDPCR | Droplet digital PCR |
GAPDH | Glyceraldehyde 3 phosphate dehydrogenase |
PMA | Phorbol 12 Myristate 13 Acetate |
SD | Standard deviations |
gRNA | guide RNA |
IDT | Integrated DNA Technologies |
HDR | Homology-directed repair |
RT qPCR | Reverse transcription quantitative polymerase chain reaction |
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Gene | Oligonucleotide Sequences |
---|---|
Guide RNA target 1 | AATTATGGAGTATGTGTCTG |
Guide RNA target 2 | ACGAGAGTAAGTAAAACTAC |
Oligo template | TGATGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAACTATGGGGTATGTTTCTGTGGAGACGAGAGTAAGTAAAACTACAGGCTTTCTAATGCCTTTCT |
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Nilsri, N.; Mekchaaum, R.; Kalasin, S.; Jongjitwimol, J.; Daowtak, K. CRISPR/Cas9-Based Modeling of JAK2 V617F Mutation in K562 Cells Reveals Enhanced Proliferation and Sensitivity to Therapeutic Agents. Int. J. Mol. Sci. 2025, 26, 4600. https://doi.org/10.3390/ijms26104600
Nilsri N, Mekchaaum R, Kalasin S, Jongjitwimol J, Daowtak K. CRISPR/Cas9-Based Modeling of JAK2 V617F Mutation in K562 Cells Reveals Enhanced Proliferation and Sensitivity to Therapeutic Agents. International Journal of Molecular Sciences. 2025; 26(10):4600. https://doi.org/10.3390/ijms26104600
Chicago/Turabian StyleNilsri, Nungruthai, Rujira Mekchaaum, Supaporn Kalasin, Jirapas Jongjitwimol, and Krai Daowtak. 2025. "CRISPR/Cas9-Based Modeling of JAK2 V617F Mutation in K562 Cells Reveals Enhanced Proliferation and Sensitivity to Therapeutic Agents" International Journal of Molecular Sciences 26, no. 10: 4600. https://doi.org/10.3390/ijms26104600
APA StyleNilsri, N., Mekchaaum, R., Kalasin, S., Jongjitwimol, J., & Daowtak, K. (2025). CRISPR/Cas9-Based Modeling of JAK2 V617F Mutation in K562 Cells Reveals Enhanced Proliferation and Sensitivity to Therapeutic Agents. International Journal of Molecular Sciences, 26(10), 4600. https://doi.org/10.3390/ijms26104600