Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells
Abstract
:1. Introduction
2. Results
2.1. CFTR Protein Rescue in the 16HBE W1282X Cell Using the REPAIRv2 System
2.2. W1282X- and G542X-Specific gRNA Design and Cloning in the mxABE Platform
2.3. Evaluation Using Fluorescence Microscopy of CFTR Protein Recovery before and after Cell Sorting
2.4. Evaluation of CFTR mRNA Levels Using RT-qPCR
3. Discussion
4. Materials and Methods
4.1. MinixABE Platform: Spacers Design and Cloning
4.2. Vectors and Clones
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- pC0055-CMV-dPspCas13b-GS-ADAR2DD(E488Q/T375G)-delta-984-1090, from Addgene (RRID: Addgene_103871);
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- pC0043-PspCas13b crRNA backbone, from Addgene (RRID: Addgene_103854);
- -
- CFTR-gRNA W1282X 50–32 and CFTR-gRNA W1282X 50-34, designed by us [22].
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- U6-BbsI-DR_CMV-minidCas13X.1-REPAIRv2-BGHpA_CMV-EGFP-BGHpA, from Addgene (RRID: Addgene_171383);
- -
- mxABE-NT, coding for a control gRNA (non-targeting) (Figure S2);
- -
- mxABE-CFW1282X 50–25, coding for a gRNA specific for the W1282X mutant region, 50 nts long, with a mismatch in position 25 with respect to 5′end (Figure S2);
- -
- mxABE-CFG542X 50–25, coding for a gRNA specific for the G542X mutant region, 50 nts long, with a mismatch in position 25 with respect to 5′end (Figure S2).
4.3. Cell Culture Conditions
4.4. Cell Transfection
4.5. RNA Extraction, cDNA Synthesis, and RT-qPCR
4.6. Immunofluorescence
4.7. Cell Sorting
4.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Chiavetta, R.F.; Titoli, S.; Barra, V.; Cancemi, P.; Melfi, R.; Di Leonardo, A. Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells. Int. J. Mol. Sci. 2023, 24, 10940. https://doi.org/10.3390/ijms241310940
Chiavetta RF, Titoli S, Barra V, Cancemi P, Melfi R, Di Leonardo A. Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells. International Journal of Molecular Sciences. 2023; 24(13):10940. https://doi.org/10.3390/ijms241310940
Chicago/Turabian StyleChiavetta, Roberta F., Simona Titoli, Viviana Barra, Patrizia Cancemi, Raffaella Melfi, and Aldo Di Leonardo. 2023. "Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells" International Journal of Molecular Sciences 24, no. 13: 10940. https://doi.org/10.3390/ijms241310940