Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors
Abstract
1. Introduction
2. Materials and Methods
2.1. Lentiviral Vector Production
2.2. In Vitro mRNA Transcription
2.3. HEK293T Cells Transfection with mRNA
2.4. Flow Cytometry and Cell Sorting
2.5. Reprogramming, hiPSC Culture and Characterization
2.6. Nucleofection
2.7. Transfection
2.8. EdU Staining of hiPSCs under Nutlin Treatment
2.9. Statistical Analysis
3. Results
3.1. Attempt for Correction of a Disease-Causing Mutation by HDR
3.2. mRNA-Mediated Base Editing in HEK293T Cells
3.3. mRNA Delivery into hiPSCs
3.4. BE-Mediated Conversion of GFP into BFP in hiPS Cells
3.5. Repair of a Disease-Causing Mutation in Patient-Derived hiPSCs
3.6. Generation of Isogenic hiPSCs with Different TP53 Mutations
3.7. Prime Editing in hiPS Cells
4. Discussion and Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sürün, D.; Schneider, A.; Mircetic, J.; Neumann, K.; Lansing, F.; Paszkowski-Rogacz, M.; Hänchen, V.; Lee-Kirsch, M.A.; Buchholz, F. Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors. Genes 2020, 11, 511. https://doi.org/10.3390/genes11050511
Sürün D, Schneider A, Mircetic J, Neumann K, Lansing F, Paszkowski-Rogacz M, Hänchen V, Lee-Kirsch MA, Buchholz F. Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors. Genes. 2020; 11(5):511. https://doi.org/10.3390/genes11050511
Chicago/Turabian StyleSürün, Duran, Aksana Schneider, Jovan Mircetic, Katrin Neumann, Felix Lansing, Maciej Paszkowski-Rogacz, Vanessa Hänchen, Min Ae Lee-Kirsch, and Frank Buchholz. 2020. "Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors" Genes 11, no. 5: 511. https://doi.org/10.3390/genes11050511
APA StyleSürün, D., Schneider, A., Mircetic, J., Neumann, K., Lansing, F., Paszkowski-Rogacz, M., Hänchen, V., Lee-Kirsch, M. A., & Buchholz, F. (2020). Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors. Genes, 11(5), 511. https://doi.org/10.3390/genes11050511