Research Article
Year 2024,
, 326 - 333, 01.10.2024
Abstract
References
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- 3. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F. RNA editing with CRISPR-Cas13. Science (1979) 358: 1019–1027, 2017. doi: 10.1126/science.aaq0180.
- 4. Huynh N, Depner N, Larson R, King-Jones K. A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila. Genome Biology 2020 21:1 21: 1–29, 2020. doi: 10.1186/S13059-020-02193-Y.
- 5. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F. RNA targeting with CRISPR–Cas13. Nature 2017 550:7675 550: 280–284, 2017. doi: 10.1038/nature24049.
- 6. Nishikura K. Functions and Regulation of RNA Editing by ADAR Deaminases. Annu Rev Biochem 79: 321–349, 2010. doi: 10.1146/annurev-biochem-060208-105251.
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- 8. Green MR, Sambrook J. Molecular cloning. A Laboratory Manual 4th 448, 2012.
- 9. Chen S, Xie W, Liu Z, Shan H, Chen M, Song Y, Yu H, Lai L, Li Z. CRISPR Start-Loss: A Novel and Practical Alternative for Gene Silencing through Base-Editing-Induced Start Codon Mutations. Mol Ther Nucleic Acids 21: 1062–1073, 2020. doi: 10.1016/J.OMTN.2020.07.037.
- 10. Zeballos C. MA, Gaj T. Next-Generation CRISPR Technologies and Their Applications in Gene and Cell Therapy. Trends Biotechnol 39: 692–705, 2021. doi: 10.1016/J.TIBTECH.2020.10.010.
- 11. Powell JE, Lim CKW, Krishnan R, McCallister TX, Saporito-Magriña C, Zeballos MA, McPheron GD, Gaj T. Targeted gene silencing in the nervous system with CRISPR-Cas13. Sci Adv 8: 2485, 2022. doi: 10.1126/sciadv.abk2485.
- 12. Sinnamon JR, Kim SY, Corson GM, Song Z, Nakai H, Adelman JP, Mandel G. Site-directed RNA repair of endogenous Mecp2 RNA in neurons. Proceedings of the National Academy of Sciences 114: E9395–E9402, 2017. doi: 10.1073/pnas.1715320114.
- 13. Fedele M, Crescenzi E, Cerchia L. The POZ/BTB and AT-Hook Containing Zinc Finger 1 (PATZ1) Transcription Regulator: Physiological Functions and Disease Involvement. Int J Mol Sci 18, 2017. doi: 10.3390/IJMS18122524.
- 14. Piepoli S, Alt AO, Atilgan C, Mancini EJ, Erman B. Structural analysis of the PATZ1 BTB domain homodimer. Acta Crystallogr D Struct Biol 76: 581–593, 2020. doi: 10.1107/S2059798320005355.
- 15. Machado I, Llombart-Bosch A, Charville GW, Navarro S, Domínguez Franjo MP, Bridge JA, Linos K. Sarcomas with EWSR1::Non-ETS Fusion (EWSR1::NFATC2 and EWSR1::PATZ1). Surg Pathol Clin 17: 31–55, 2024. doi: 10.1016/J.PATH.2023.07.001.
Designing and Cloning Guide RNA Plasmids for Targeted Editing of Mammalian RNAs by Using CRISPR-Cas13b
Abstract
Background/Purpose: CRISPR/Cas13 expands the CRISPR/Cas9-mediated DNA editing approaches to the RNA editing. In this system, a guide RNA (gRNA) targets a specific region in the RNA of interest and recruits Cas13. gRNA can be designed with little restriction to cover almost the whole transcriptome and various engineered Cas13 enzymes with unique added features can be utilized at the region of interest.
Methods: Plasmids were obtained from Addgene plasmid repository and their integrities were first confirmed by restriction enzyme digestions. An oligo that is complementary to the region surrounding the start codon of PATZ1 mRNA was designed and cloned into a gRNA plasmid by using golden gate reaction. The cloned plasmid was confirmed by Sanger-sequencing.
Results: A 45-nucleotide long sequence that is complementary to PATZ1 mRNA around the AUG start codon with a mismatched cytosine for the corresponding adenine at the 30th nucleotide from the 3’ end of the sequence was designed and cloned into gRNA plasmid.
Conclusion: We designed and cloned a gRNA plasmid that targets the start codon of human PATZ1 mRNA. When this plasmid is co-transfected into cells with a catalytically inactivated Cas13 fused to an adenosine deaminase encoding plasmid, the adenine nucleotide in the canonical start codon of PATZ1 is expected to be edited to inosine. This change might be functionally important to study the decrease in protein translation or the truncation of N-termini in future studies.
Keywords: CRISPR-Cas Systems, RNA editing, ADAR Protein, PATZ1 protein
References
- 1. Devillars A, Magon G, Pirrello C, Palumbo F, Farinati S, Barcaccia G, Lucchin M, Vannozzi A. Not Only Editing: A Cas-Cade of CRISPR/Cas-Based Tools for Functional Genomics in Plants and Animals. International Journal of Molecular Sciences 2024, Vol 25, Page 3271 25: 3271, 2024. doi: 10.3390/IJMS25063271.
- 2. Cebrailoglu N, Yildiz AB, Akkaya O, Ciftci YO. CRISPR-Cas: Removing Boundaries of the Nature. European Journal of Biology 78: 157–164, 2019. doi: 10.26650/EurJBiol.2019.0024.
- 3. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F. RNA editing with CRISPR-Cas13. Science (1979) 358: 1019–1027, 2017. doi: 10.1126/science.aaq0180.
- 4. Huynh N, Depner N, Larson R, King-Jones K. A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila. Genome Biology 2020 21:1 21: 1–29, 2020. doi: 10.1186/S13059-020-02193-Y.
- 5. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F. RNA targeting with CRISPR–Cas13. Nature 2017 550:7675 550: 280–284, 2017. doi: 10.1038/nature24049.
- 6. Nishikura K. Functions and Regulation of RNA Editing by ADAR Deaminases. Annu Rev Biochem 79: 321–349, 2010. doi: 10.1146/annurev-biochem-060208-105251.
- 7. Keskin N, Deniz E, Eryilmaz J, Un M, Batur T, Ersahin T, Atalay RC, Sakaguchi S, Ellmeier W, Erman B. PATZ1 Is a DNA Damage-Responsive Transcription Factor That Inhibits p53 Function. Mol Cell Biol 35: 1741–1753, 2015. doi: 10.1128/MCB.01475-14.
- 8. Green MR, Sambrook J. Molecular cloning. A Laboratory Manual 4th 448, 2012.
- 9. Chen S, Xie W, Liu Z, Shan H, Chen M, Song Y, Yu H, Lai L, Li Z. CRISPR Start-Loss: A Novel and Practical Alternative for Gene Silencing through Base-Editing-Induced Start Codon Mutations. Mol Ther Nucleic Acids 21: 1062–1073, 2020. doi: 10.1016/J.OMTN.2020.07.037.
- 10. Zeballos C. MA, Gaj T. Next-Generation CRISPR Technologies and Their Applications in Gene and Cell Therapy. Trends Biotechnol 39: 692–705, 2021. doi: 10.1016/J.TIBTECH.2020.10.010.
- 11. Powell JE, Lim CKW, Krishnan R, McCallister TX, Saporito-Magriña C, Zeballos MA, McPheron GD, Gaj T. Targeted gene silencing in the nervous system with CRISPR-Cas13. Sci Adv 8: 2485, 2022. doi: 10.1126/sciadv.abk2485.
- 12. Sinnamon JR, Kim SY, Corson GM, Song Z, Nakai H, Adelman JP, Mandel G. Site-directed RNA repair of endogenous Mecp2 RNA in neurons. Proceedings of the National Academy of Sciences 114: E9395–E9402, 2017. doi: 10.1073/pnas.1715320114.
- 13. Fedele M, Crescenzi E, Cerchia L. The POZ/BTB and AT-Hook Containing Zinc Finger 1 (PATZ1) Transcription Regulator: Physiological Functions and Disease Involvement. Int J Mol Sci 18, 2017. doi: 10.3390/IJMS18122524.
- 14. Piepoli S, Alt AO, Atilgan C, Mancini EJ, Erman B. Structural analysis of the PATZ1 BTB domain homodimer. Acta Crystallogr D Struct Biol 76: 581–593, 2020. doi: 10.1107/S2059798320005355.
- 15. Machado I, Llombart-Bosch A, Charville GW, Navarro S, Domínguez Franjo MP, Bridge JA, Linos K. Sarcomas with EWSR1::Non-ETS Fusion (EWSR1::NFATC2 and EWSR1::PATZ1). Surg Pathol Clin 17: 31–55, 2024. doi: 10.1016/J.PATH.2023.07.001.
There are 15 citations in total.
Details
| Primary Language | English |
|---|---|
| Subjects | Biochemistry and Cell Biology (Other) |
| Journal Section | Research Articles |
| Authors | |
| Early Pub Date | September 17, 2024 |
| Publication Date | October 1, 2024 |
| Submission Date | April 9, 2024 |
| Acceptance Date | June 10, 2024 |
| Published in Issue | Year 2024 |