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

Abstract

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.

Mapping the landscape of HPV integration and characterising virus and host genome interactions in HPV‐positive oropharyngeal squamous cell carcinoma

“…Additionally, noteworthy progress has been made in gene editing therapies targeting HPV oncogene E6 and E7 in Hela cells, leading to the up-regulation of tumour suppressor proteins p53/Rb. 60 Therefore, gene editing technologies hold promise for targeting genomic sites with viral integration or cells harbouring viral insertions. As highlighted earlier, HPV integration may lead to dysregulation of oncogenes, exerting carcinogenic effects independently of E6 and E7.…”

“…In the context of cervical cancer, ongoing clinical trials are investigating the significance of HPV integration status in screening approaches, 59 offering valuable insightful for potential screening strategies in HPV(+)OPSCC. Additionally, noteworthy progress has been made in gene editing therapies targeting HPV oncogene E6 and E7 in Hela cells, leading to the up‐regulation of tumour suppressor proteins p53/Rb 60 . Therefore, gene editing technologies hold promise for targeting genomic sites with viral integration or cells harbouring viral insertions.…”

CRISPR-Cas System: The Current and Emerging Translational Landscape

“…The status of this study is unknown and was last updated in 2017. In the interim, the team has been publishing results from their efforts to improve editing efficiency of the HPV E6/E7 oncogenes through a method called gene knock-out chain reaction (PMID: 35036522) [ 133 ], and exploration of a CRISPR/Cas13a system to knock-out HPV E6/E7 mRNAs in vivo cell culture [ 134 ].…”

Description of CRISPR-Cas9 development and its prospects in human papillomavirus-driven cancer treatment

“…However, different HPV genotypes of HPV-driven cancers, such as cervical cancer patients with HPV-18 DNA, have significantly different responses to radiotherapy ( 89 ). Recently, a novel screen based on a targeted CRISPR−Cas9 system was applied to identify optimal radiosensitization approaches for HPV-positive/negative HNSCC ( 90 ). The combination of radiotherapy and CRISPR−Cas9-mediated inhibition of genetic repair pathways could improve the therapeutic response in patients with HNSCC.…”