Construction of novel gRNA CRSIPR-Cas 9 vectors against HPV 18 E6 and E7 oncoproteins

Abstract

High risk human papilloma viruses are the main etiological agents of cervical cancers responsible for approximately 99.7% of all cases. The persistence of viral infection is the major reason that triggers oncogenesis in host’s infected cells due to the deregulation of oncogenes E6 and E7. The current treatment regimens include cisplatin base chemotherapy (CRT), radiotherapy and hysterectomy, however, these approaches have their downfalls as CRT has a recurrence rate of 25-40% and surgery results in loss of fertility. CRISPR-Cas9 therapy can prove to be an effective approach to curb viral persistence targeting HPV oncogenes at genetic level and can be an effective remedy for cervical cancer. This study employs the use of CRISPR-Cas9 gene editing system to target E6 and E7 genes of HPV-18 virus. Novel guide RNAs against oncogenes E6 and E7 genes of HPV-18 were designed and their off-targets in human genome were evaluated of CasOFFinder software. They were then cloned in pX260 CRISPR Cas 9 expression vector. The confirmation of cloning was done by sangar sequencing. The resultant E6 gRNA-CRISPR Cas9 and E7 gRNA-CRISPR Cas9 vectors can be potentially therapeutic against HPV-18 after their efficacy is evaluated in vitro and in vivo.