Journal of Advances in Medical and Biomedical Research

en Developing a CRISPR/Cas9 Plasmid Vector Containing Specific Single Guide RNAs Targeting SMO in the Sonic Hedgehog Signaling Pathway for SHH-Driven Cancers Medical Biology Medical Biology مقاله پژوهشی Original Research Article 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. CRISPR/Cas9, Plasmids/Genetics, Vector, Hedgehog Proteins/ Genetics, Cancer 23 30 http://journal.zums.ac.ir/browse.php?a_code=A-10-2691-6&slc_lang=en&sid=1 Mehrdad Ghorbanlou mehrdad.ghorbanlou@gmail.com 5200319475328460088265 5200319475328460088265 No Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Ali Salimi-Jeda salimi.ali84@gmail.com 5200319475328460088266 5200319475328460088266 No Applied Virology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran Mohamad Reza Razavi mrrazavi@yahoo.com 5200319475328460088267 5200319475328460088267 No Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran Ronak Shabani shabanironak@gmail.com 5200319475328460088268 5200319475328460088268 No Reproductive Sciences and Technology Research Center, Department of Anatomy, Iran University of Medical Sciences, Tehran, Iran Fatemeh Moradi f7moradi@gmail.com 5200319475328460088269 5200319475328460088269 No Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Hassan Marzban hassan.marzban@umanitoba.ca 5200319475328460088270 5200319475328460088270 No Department of Human Anatomy and Cell Science, Winnipeg, MB, Canada Mehdi Mehdizadeh mehdizadeh.m@iums.ac.ir 5200319475328460088271 5200319475328460088271 Yes Reproductive Sciences and Technology Research Center, Department of Anatomy, Iran University of Medical Sciences, Tehran, Iran