Genome-Wide CRISPR Screen Identifies KEAP1 Perturbation as a Vulnerability of ARID1A-Deficient Cells
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Genome-Wide CRISPR Screen Identifies Genes Important for Fitness in ARID1A-KO CCOC Cells
2.2. Validation of KEAP1 as a Fitness Gene in ARID1A-Deficient Cells
2.3. Gene Expression Analysis Reveals a Dysregulation of NRF2 Target Genes in ARID1A-KO Cells
2.4. Aberrant NRF2 Signalling Does Not Significantly Impair the Growth of ARID1A-KO Cells
2.5. KEAP1 Perturbation Exacerbates Genome Instability in ARID1A-Deficient Cells
2.6. Dual Inhibition of KEAP1 and ATR Potentiates Killing of ARID1A-KO Cells
3. Discussion
4. Methods
4.1. Cell Culture Conditions
4.2. Generation of NRF2E79Q Dox-Inducible Cell Lines
4.3. TKOv3 Library Cloning and Viral Preparation
4.4. TKOv3 Library Viral Transduction and Titration
4.5. CRISPR Knockout Pool Generation
4.6. Genomic DNA Extraction and Precipitation
4.7. Library Preparation and Sequencing
4.8. Amplicon Scoring, Guide-RNA Enrichment and Hit Identification
4.9. Gene Ontology (GO) Analysis
4.10. Incucyte Growth Assay
4.11. RNA Interference
4.12. Crystal Violet Proliferation Assay
4.13. Immunofluorescence
4.14. Western Blotting
4.15. TCGA Dataset Analysis
4.16. Differential Gene Expression Analysis
4.17. Culture of Patient-Derived Endometrial Progenitor Cells
4.18. Statistical Analysis, Data Availability, and Reproducibility
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Fournier, L.-A.; Kalantari, F.; Wells, J.P.; Lee, J.S.; Trigo-Gonzalez, G.; Moksa, M.M.; Smith, T.; White, J.; Shanks, A.; Wang, S.L.; et al. Genome-Wide CRISPR Screen Identifies KEAP1 Perturbation as a Vulnerability of ARID1A-Deficient Cells. Cancers 2024, 16, 2949. https://doi.org/10.3390/cancers16172949
Fournier L-A, Kalantari F, Wells JP, Lee JS, Trigo-Gonzalez G, Moksa MM, Smith T, White J, Shanks A, Wang SL, et al. Genome-Wide CRISPR Screen Identifies KEAP1 Perturbation as a Vulnerability of ARID1A-Deficient Cells. Cancers. 2024; 16(17):2949. https://doi.org/10.3390/cancers16172949
Chicago/Turabian StyleFournier, Louis-Alexandre, Forouh Kalantari, James P. Wells, Joon Seon Lee, Genny Trigo-Gonzalez, Michelle M. Moksa, Theodore Smith, Justin White, Alynn Shanks, Siyun L. Wang, and et al. 2024. "Genome-Wide CRISPR Screen Identifies KEAP1 Perturbation as a Vulnerability of ARID1A-Deficient Cells" Cancers 16, no. 17: 2949. https://doi.org/10.3390/cancers16172949