• Cloning of a novel designed guide RNA into the CRISPR/Cas9 vector for knocking out HPV16 E6 oncogene
  • Niloofar Khairkhah,1 Azam Bolhassani,2,* Reza Najafipour,3
    1. Department of Molecular Medicine, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran
    2. Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
    3. Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran


  • Introduction: Human papillomavirus (HPV) is the most common sexually transmitted virus in the world. About 70% of cervical cancers are caused by the most oncogenic HPV genotypes of 16 and 18. Since available prophylactic vaccines do not induce immunity in those with established HPV infections, the development of a therapeutic approach remains essential. CRISPR/Cas9 system is a genome-editing method that can be considered as a therapeutic approach by knocking out the viral oncogenes resulting in tumor growth suppression. In this study, we aimed to clone a novel designed guide RNA into the PX330 CRISPR/Cas9 all-in-one vector since this part of the study is considered a significant bottleneck.
  • Methods: PX330 all-in-one CRISPR/Cas9 vector had been purchased from Zhang’s lab. E6 Target site of HPV16 were selected by utilizing CHOPCHOP online tool. one single-stranded oligo was ordered in the form of incompatible overhang for correct orientation and cloned into the plasmid using BbsI restriction enzyme. The final confirmation of the cloned guide RNA oligo was conducted by Sanger sequencing.
  • Results: The final confirmation by Sanger sequencing showed that the designed oligo was successfully cloned into the px330 all-in-one CRISPR/Cas9 vector.
  • Conclusion: The CRISPR/Cas9 system has demonstrated high-fidelity gene editing and has become widely used for specific targeting and cleavage. This is a viable route of treatment that could supplement or potentially replace the current treatments of surgery, chemo and radiation therapy.
  • Keywords: human papillomavirus; cervical cancer; CRISPR/Cas9