Gene knock-out chain reaction enables high disruption efficiency of HPV18 E6/E7 genes in cervical cancer cells

• DOI: 10.1016/j.omto.2021.12.011
• Publication Date: 2021-12-18T03:51:58Z
• Authors: Rui Tian; Jiashuo Liu; Weiwen Fan; Rui Li; Zifeng Cui; Zhuang Jin; Zhaoyue Huang; Hongxian Xie; Lifang Li; Zheying Huang; Zheng Hu; Ping Zhou; Xun Tian
• Keywords: 0301 basic medicine; HPV; 03 medical and health sciences; cervical cancer; Neoplasms. Tumors. Oncology. Including cancer and carcinogens; Original Article; CRISPR-Cas9; gene knockout chain reaction (GKCR); HITI; RC254-282; 3. Good health

A genome editing tool targeting the high-risk human papillomavirus (HPV) oncogene is a promising therapeutic strategy to treat HPV-related cervical cancer. To improve gene knockout efficiency, we developed a gene knockout chain reaction (GKCR) method for continually generating mutagenic disruptions and used this method to disrupt the HPV18 E6 and E7 genes. We verified that the GKCR Cas9/guide RNA (gRNA) cassettes could integrated into the targeted loci via homology-independent targeted insertion (HITI). The qPCR results revealed that the GKCR method enabled a relatively higher Cas9/gRNA cassette insertion rate than a control method (the common CRISPR-Cas9 strategy). Tracking of Indels by DEcomposition (TIDE) assay results showed that the GKCR method produced a significantly higher percentage of insertions or deletions (indels) in the HPV18 E6 and E7 genes. Furthermore, by targeting the HPV18 E6/E7 oncogenes, we found that the GKCR method significantly upregulated the P53/RB proteins and inhibited the proliferation and motility of HeLa cells. The GKCR method significantly improved the gene knockout efficiency of the HPV18 E6/E7 oncogenes, which might provide new insights into treatment of HPV infection and related cervical cancer.