Volume 34, Issue 1 (January & February 2026)                   J Adv Med Biomed Res 2026, 34(1): 23-30 | Back to browse issues page

Ethics code: IR.IUMS.FMD.REC.1399.543

XML Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Ghorbanlou M, Salimi-Jeda A, Razavi M R, Shabani R, Moradi F, Marzban H et al . Developing a CRISPR/Cas9 Plasmid Vector Containing Specific Single Guide RNAs Targeting SMO in the Sonic Hedgehog Signaling Pathway for SHH-Driven Cancers. J Adv Med Biomed Res 2026; 34 (1) :23-30
URL: http://journal.zums.ac.ir/article-1-7723-en.html
Developing a CRISPR/Cas9 Plasmid Vector Containing Specific Single Guide RNAs Targeting SMO in the Sonic Hedgehog Signaling Pathway for SHH-Driven Cancers
Mehrdad Ghorbanlou1 , Ali Salimi-Jeda2 , Mohamad Reza Razavi3 , Ronak Shabani4 , Fatemeh Moradi1 , Hassan Marzban5 , Mehdi Mehdizadeh *6
1- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
2- Applied Virology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
3- Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran
4- Reproductive Sciences and Technology Research Center, Department of Anatomy, Iran University of Medical Sciences, Tehran, Iran
5- Department of Human Anatomy and Cell Science, Winnipeg, MB, Canada & Children’s Hospital Research Institute of Manitoba (CHRIM), College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada
6- Reproductive Sciences and Technology Research Center, Department of Anatomy, Iran University of Medical Sciences, Tehran, Iran , mehdizadeh.m@iums.ac.ir
Abstract:   (92 Views)

Background & Objective: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is considered an efficient tool for genomic engineering in the field of cancer/gene therapy. Developing a plasmid vector to target the signaling pathway of sonic hedgehog (SHH), a key driver of malignancies such as medulloblastoma and basal cell carcinoma, is the aim of this study.
 Materials & Methods: CRISPOR online platform was used to select three specific and efficient gRNAs (guide RNAs) to design and then synthesize a gRNA cassette in a plasmid vector to target SHH pathway. Following digestion of pCas-guide-EF1a-GFP, the Cas9 expressing vector, by BsrGI and BamHI restriction enzymes and ligation with T4 ligase, the gRNA cassette was cloned. After that, competent DH5α E. coli bacteria were used to amplificate the vector. Finally, after plasmid extraction, presence of the gRNA cassette was confirmed through DNA sequencing and polymerase chain reaction (PCR).
Results:  To target SMO gene, as an upstream target of SHH pathway, three target-prone exons (2nd, 4th, and 6th) were used to synthesize efficient and specific gRNAs. The process of cloning and developing the interested vector was confirmed by sequencing and PCR.
Conclusion:  Although this study lacks in vitro/in vivo validation of the candidate gRNAs within the plasmid vector, developing efficient, and specific CRISPR/Cas9 vectors may possibly be a promising approach in the field of cancer treatment through targeting neoplastic pathways at the level of positive regulators which may possibly lead to a better perception of different molecular pathways leading to cancer, and developing precise chemotherapeutic agents.

Keywords: CRISPR/Cas9, Plasmids/Genetics, Vector, Hedgehog Proteins/ Genetics, Cancer
Full-Text [PDF 1051 kb]   (68 Downloads)    
Type of Study: Original Research Article | Subject: Medical Biology
Received: 2025/07/28 | Accepted: 2026/02/8 | Published: 2026/02/28
References
1. Asmamaw M, Zawdie B. Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing. Biologics. 2021;2021(15):353-61. [DOI:10.2147/BTT.S326422] [PMID] [PMCID]
2. El-Mounadi K, Morales-Floriano M, Garcia-Ruiz H. Principles, Applications, and Biosafety of Plant Genome Editing Using CRISPR-Cas9. Front Plant Sci. 2020;11. [DOI:10.3389/fpls.2020.00056] [PMID] [PMCID]
3. Xue C, Greene E. DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing. Trends Genet. 2021;37(7):639-56. [DOI:10.1016/j.tig.2021.02.008] [PMID] [PMCID]
4. Liu Z, Shi M, Ren Y, Xu H, Weng S, Ning W, et al. Recent advances and applications of CRISPR-Cas9 in cancer immunotherapy. Mol Cancer. 2023;22(1):35. https://doi.org/10.1186/s12943-023-01738-6 https://doi.org/10.1186/1476-4598-6-35 [DOI:10.1186/s12943-024-01950-y]
5. Palla M, Scarpato L, Trolio R, Ascierto P. Sonic hedgehog pathway for the treatment of inflammatory diseases: implications and opportunities for future research. J Immunother Cancer. 2022;10(6):e004397. [DOI:10.1136/jitc-2021-004397] [PMID] [PMCID]
6. Smelkinson M. The Hedgehog signaling pathway emerges as a pathogenic target. J Dev Biol. 2017;5(4):14. [DOI:10.3390/jdb5040014] [PMID]
7. Wang W, Shiraishi R, Kawauchi D. Sonic Hedgehog Signaling in Cerebellar Development and Cancer. Front Cell Dev Biol. 2022;2022(29):10. [DOI:10.3389/fcell.2022.864035] [PMID] [PMCID]
8. Gambini D, Passoni E, Nazzaro G, Beltramini G, Tomasello G, Ghidini M, et al. Basal Cell Carcinoma and Hedgehog Pathway Inhibitors: Focus on Immune Response. Front Med. 2022; 2022(14):9. [DOI:10.3389/fmed.2022.893063] [PMID] [PMCID]
9. Skoda A, Simovic D, Karin V, Kardum V, Vranic S, Serman L. The role of the Hedgehog signaling pathway in cancer: A comprehensive review. Bosn J Basic Med Sci. 2018;18(1):8-20. [DOI:10.17305/bjbms.2018.2756] [PMID] [PMCID]
10. Sigafoos A, Paradise B, Fernandez-Zapico M. Hedgehog/GLI Signaling Pathway: Transduction, Regulation, and Implications for Disease. Cancers (Basel). 2021;13(14). [DOI:10.3390/cancers13143410] [PMID] [PMCID]
11. Behruzi M, Ghorbanlou M, Shabani R, Rustamzadeh A, Alhagh Gorgich E, Seidkhani E, et al. Vismodegib Improved Therapy of Medulloblastoma by Targeting Sonic Hedgehog Signaling Pathway on DAOY Medulloblastoma Cell Line. Basic Clin Neurosci J. 2025;16(3):609-18. [DOI:10.32598/bcn.2024.6423.1]
12. Amakye D, Jagani Z, Dorsch M. Unraveling the therapeutic potential of the Hedgehog pathway in cancer. Nat Med. 2013;19(11):1410-22. [DOI:10.1038/nm.3389] [PMID]
13. Concordet J-P, Haeussler M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018;46(W1):W242-W5. [DOI:10.1093/nar/gky354] [PMID] [PMCID]
14. Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J, Renaud J-B, et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 2016;17(1):148. [DOI:10.1186/s13059-016-1012-2] [PMID] [PMCID]
15. Wu F, Gao G, Pan T, Yang Z, Xu G, Abbas N, et al. Generation of a SMO homozygous knockout human embryonic stem cell line WAe001-A-16 by CRISPR/Cas9 editing. Stem Cell Research. 2018;2018(27):5-9. [DOI:10.1016/j.scr.2017.12.002] [PMID]
16. Singh M, Yadav A, Ma X, Amoah E. Plasmid DNA transformation in Escherichia Coli: effect of heat shock temperature, duration, and cold incubation of CaCl2 treated cells. Int J Biotechnol Biochem. 2010;2010(6):561-8.
17. Javadi A, Shamaei M, Mohammadi Ziazi L, Pourabdollah M, Dorudinia A, Seyedmehdi S, et al. Qualification study of two genomic DNA extraction methods in different clinical samples. Tanaffos. 2014;13(4):41-7.
18. Sung H, Ferlay J, Siegel R, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49. [DOI:10.3322/caac.21660] [PMID]
19. Chen X-Z, Guo R, Zhao C, Xu J, Song H, Yu H, et al. A Novel Anti-Cancer Therapy: CRISPR/Cas9 Gene Editing. Front Pharmacol. 2022(22):13. [DOI:10.3389/fphar.2022.939090] [PMID] [PMCID]
20. Mun E, Babiker H, Weinberg U, Kirson E, Von Hoff D. Tumor-Treating Fields: A Fourth Modality in Cancer Treatment. Clin Cancer Res. 2018;24(2):266-75. [DOI:10.1158/1078-0432.CCR-17-1117] [PMID] [PMCID]
21. Vanneman M, Dranoff G. Combining immunotherapy and targeted therapies in cancer treatment. Nat Rev Cancer. 2012;12(4): 237-51. [DOI:10.1038/nrc3237] [PMID] [PMCID]
22. Ghorbanlou M, Marzban H, Mehdizadeh M. Targeting Positive Regulators of Sonic Hedgehog Signaling Pathway in Medulloblastoma by Designing CRISPR/Cas9 Single Guide RNAs. J Adv Med Biomed Res. 2023;31(149):594-601. [DOI:10.30699/jambs.31.149.594]
23. Ghorbanlou M, Moradi F, Shabani R, Mehdizadeh M. Upregulation of apoptotic genes and downregulation of target genes of Sonic Hedgehog signaling pathway in DAOY medulloblastoma cell line treated with arsenic trioxide. J Chemother. 2024;36(6):506-19. [DOI:10.1080/1120009X.2023.2294574] [PMID]
24. Gampala S, Zhang G, Chang C, Yang J-Y. Activation of AMPK sensitizes medulloblastoma to Vismodegib and overcomes Vismodegib-resistance. FASEB BioAdv. 2021;3(6):459-69. [DOI:10.1096/fba.2020-00032] [PMID] [PMCID]
25. Li Y, Song Q, Day B. Phase I and phase II sonidegib and vismodegib clinical trials for the treatment of paediatric and adult MB patients: a systemic review and meta-analysis. Acta Neuropathol Commun. 2019;7(1):123. [DOI:10.1186/s40478-019-0773-8] [PMID] [PMCID]
26. Lou E, Schomaker M, Wilson J, Ahrens M, Dolan M, Nelson A. Complete and sustained response of adult medulloblastoma to first-line sonic hedgehog inhibition with vismodegib. Cancer Biol Ther. 2016;17(10):1010-6. [DOI:10.1080/15384047.2016.1220453] [PMID] [PMCID]
27. Kim J, Aftab B, Tang J, Kim D, Lee A, Rezaee M, et al. Itraconazole and arsenic trioxide inhibit Hedgehog pathway activation and tumor growth associated with acquired resistance to smoothened antagonists. Cancer Cell. 2013; 23(1):23-34. [DOI:10.1016/j.ccr.2012.11.017] [PMID] [PMCID]
28. Sekulic A, Migden M, Oro A, Dirix L, Lewis K, Hainsworth J, et al. Efficacy and safety of vismodegib in advanced basal-cell carcinoma. N Engl J Med. 2012;366(23):2171-9. [DOI:10.1056/NEJMoa1113713] [PMID] [PMCID]
29. Yauch R, Dijkgraaf G, Alicke B, Januario T, Ahn C, Holcomb T, et al. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science. 2009 326(5952):572-4. [DOI:10.1126/science.1179386] [PMID] [PMCID]
30. Buonamici S, Williams J, Morrissey M, Wang A, Guo R, Vattay A, et al. Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma. Sci Transl Med. 2010;2(51): 51ra70. [DOI:10.1126/scitranslmed.3001599] [PMID] [PMCID]
31. Baliou S, Adamaki M, Kyriakopoulos A, Spandidos D, Panayiotidis M, Christodoulou I, et al. CRISPR therapeutic tools for complex genetic disorders and cancer (Review). Int J Oncol Aug. 2018;53(2):443-68. [DOI:10.3892/ijo.2018.4434] [PMID] [PMCID]
32. Liu G, Zhang Y, Zhang T. Computational approaches for effective CRISPR guide RNA design and evaluation. Comput Struct Biotechnol J. 2020;2020(18):35-44. [DOI:10.1016/j.csbj.2019.11.006] [PMID] [PMCID]
33. Yousefi-Najafabadi Z, Mehmandoostli Z, Asgari Y, Kaboli S, Falak R, Kardar G. Reversing T Cell Exhaustion by Converting Membrane PD-1 to Its Soluble form in Jurkat Cells; Applying The CRISPR/Cas9 Exon Skipping Strategy. Cell Journal (Yakhteh). 2023;25(9):633-44.
34. Shamsara M, Jamshidizad A, Rahim-Tayefeh A, Davari M, Rajabi Zangi A, Masoumi F, et al. Generation of Mouse Model of Hemophilia A by Introducing Novel Mutations, Using CRISPR/Nickase Gene Targeting System. Cell J (Yakhteh). 2023;25(9):655-9.
35. Salimi-Jeda A, Esghaei M, Hossein k, Bokharaei-Salim F, Teimoori A. Inhibition of HIV-1 replication using the CRISPR/cas9-no NLS system as a prophylactic strategy. Heliyon. 2022;8(9):e10483. [DOI:10.1016/j.heliyon.2022.e10483] [PMID] [PMCID]
36. Du Y, Liu Y, Hu J, Peng X. CRISPR/Cas9 systems: Delivery technologies and biomedical applications. Asian J Pharm Sci. 2023;18(6): 100854. [DOI:10.1016/j.ajps.2023.100854] [PMID] [PMCID]
37. Doench J, Fusi N, Sullender M, Hegde M, Vaimberg E, Donovan K, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34(2):184-91. [DOI:10.1038/nbt.3437] [PMID] [PMCID]
38. Naeem M, Majeed S, Hoque M. Latest developed strategies to minimize the off-target effects in CRISPR-Cas-mediated genome editing. Cells. 2020;9(7):1608. [DOI:10.3390/cells9071608] [PMID] [PMCID]
39. Richter M, Zhao K, Eton E, Lapinaite A, Newby G, Thuronyi B, et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol. 2020;38(7):883-91. https://doi.org/10.1038/s41587-020-0453-z [DOI:10.1038/s41587-020-0562-8] [PMID] [PMCID]
40. Scott M, Hu Y. Generation of CRISPR-Edited Rodents Using a Piezo-Driven Zygote Injection Technique. Methods Mol Biol. 2019(1874): 169-78. [DOI:10.1007/978-1-4939-8831-0_9] [PMID]
41. Wang H, Li M, Lee C, Chakraborty S, Kim H, Bao G, et al. CRISPR/Cas9-Based Genome Editing for Disease Modeling and Therapy: Challenges and Opportunities for Nonviral Delivery. Chem Rev. 2017;117(15):9874-906. [DOI:10.1021/acs.chemrev.6b00799] [PMID] [PMCID]
42. Liu Z, Liao Z, Chen Y, Han L, Yin Q, Xiao H. Application of various delivery methods for CRISPR/dCas9. Mol Biotechnol. 2020;62(8): 355-63. [DOI:10.1007/s12033-020-00258-8] [PMID]
43. Rani V, Prabhu A. CRISPR-Cas9 based non-viral approaches in nanoparticle elicited therapeutic delivery. J Drug Deliv Sci Technol. 2022(76):103737. [DOI:10.1016/j.jddst.2022.103737]
Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2026 CC BY-NC 4.0 | Journal of Advances in Medical and Biomedical Research

Designed & Developed by : Yektaweb