• Developing a CRISPR/Cas9 plasmid vector containing specific single guide RNAs targeting sonic hedgehog signaling pathway: an anti-cancer approach
  • Mehrdad Ghorbanlou,1 Ali Salimi-Jeda,2 Mohamad Reza Razavi,3 Ronak Shabani,4 Fatemeh Moradi,5 Mehdi Mehdizadeh,6,*
    1. Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
    2. Department of Virology, School of Medicine, Iran 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 Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
    6. Reproductive Sciences and Technology Research Center, Department of Anatomy, Iran University of Medical Sciences, Tehran, Iran


  • Introduction: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is an effective genomic engineering tool applied in gene/cancer therapy. This study aims to develop a vector for efficient targeting of sonic hedgehog (SHH) signaling pathway.
  • Methods: For developing the SHH-targeting plasmid vector, a gRNA (guide RNA) cassette containing three efficient and specific gRNAs was designed by CRISPOR online platform and synthesized. Then it was cloned into the Cas9 expressing plasmid vector, pCas-guide-EF1a-GFP, following digestion by BamHI and BsrGI restriction enzymes, and ligation with T4 ligase. Then the ligation mixture was chemically transformed and proliferated in competent E.coli DH5α. Following plasmid extraction, the polymerase chain reaction (PCR), and DNA sequencing was performed to confirm the presence of gRNA cassette.
  • Results: Three specific, and efficient gRNAs for second, fourth, and sixth exons of SMO gene were carefully selected and successfully synthesized. Cloning of the gRNA cassette into the pCas-guide-EF1a-GFP vector was confirmed by PCR, and sequencing of the gRNA cassette.
  • Conclusion: Developing specific, and efficient CRISPR/Cas9 vectors to target positive regulators of neoplastic pathways is a promising approach in anti-cancer target therapy which may lead to better understanding of various molecular pathways leading to cancer, and developing meticulous chemotherapy agents.
  • Keywords: CRISPR/Cas9, plasmid, vector, SHH, cancer