Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma
Abstract
:1. Introduction
2. Results
2.1. Dual sgRNA CRISPR/Cas9-Mediated Strategy to Target LRP1B
2.2. Evaluation of LRP1B Alterations in Single-Cell-Derived Clones from CRISPR/Cas9-Transfected Cells
2.3. Evaluation of LRP1B Silencing in Cell Clones
2.4. Effect of LRP1B Alterations on Tumor Growth In Vitro and In Vivo
2.5. Effect of LRP1B Alterations on Cell Secretome
3. Discussion
4. Materials and Methods
4.1. LRP1B Target Region
4.2. CRISPR/Cas9 Expression Vector
4.3. sgRNA Oligonucleotide Duplex Preparation and Cloning into PX459 Vector
4.4. Cell Culture
4.5. Cell Transfection
4.6. Genomic DNA Extraction, PCR, and Sanger Sequencing Analysis
4.7. RNA Extraction and LRP1B mRNA Expression Analysis
4.8. Cell Growth and Cell Cycle Profile Analysis
4.9. Immunofluorescence Analysis
4.10. CAM Assay
4.11. Collection of Secretome
4.12. Proteomic Evaluation Using Liquid Chromatography–Mass Spectrometry
4.13. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Target Exon | Clone | Deletion Length (bp) | Predicted Effect on LRP1B mRNA |
---|---|---|---|
1 | B9 | 94 (precise deletion) | Loss of the putative Kozac consensus sequence a and the canonical AUG start codon |
93 (1 bp insertion) | Loss of the putative Kozac consensus sequence a and the canonical AUG start codon | ||
E6 | 26 (1 bp deletion in sgRNA1-binding region) and 25 bp deletion in the sgRNA2-binding region) | Loss of the short consensus sequence at exon 1–intron 1 junction (5′ splice site) | |
H7 | 92 (2 bp insertion) | Loss of the putative Kozac consensus sequence a and the canonical AUG start codon | |
84 (20 bp insertion) | Preservation of the putative Kozac consensus sequence a and the canonical AUG start codon | ||
85 | B9 | No deletion | NA |
E6 | 2 bp deletion in the sgRNA4-binding region | Introduction of an ORF-interrupting PTC | |
H7 | 1 bp deletion in the sgRNA4-binding region2 bp deletion in the sgRNA4-binding region | Introduction of an ORF-interrupting PTC |
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Peixoto, J.; Príncipe, C.; Pestana, A.; Osório, H.; Pinto, M.T.; Prazeres, H.; Soares, P.; Lima, R.T. Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma. Int. J. Mol. Sci. 2023, 24, 11285. https://doi.org/10.3390/ijms241411285
Peixoto J, Príncipe C, Pestana A, Osório H, Pinto MT, Prazeres H, Soares P, Lima RT. Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma. International Journal of Molecular Sciences. 2023; 24(14):11285. https://doi.org/10.3390/ijms241411285
Chicago/Turabian StylePeixoto, Joana, Catarina Príncipe, Ana Pestana, Hugo Osório, Marta Teixeira Pinto, Hugo Prazeres, Paula Soares, and Raquel T. Lima. 2023. "Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma" International Journal of Molecular Sciences 24, no. 14: 11285. https://doi.org/10.3390/ijms241411285
APA StylePeixoto, J., Príncipe, C., Pestana, A., Osório, H., Pinto, M. T., Prazeres, H., Soares, P., & Lima, R. T. (2023). Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma. International Journal of Molecular Sciences, 24(14), 11285. https://doi.org/10.3390/ijms241411285