Journal
MOLECULAR THERAPY-ONCOLYTICS
Volume 24, Issue -, Pages 171-179Publisher
CELL PRESS
DOI: 10.1016/j.omto.2021.12.011
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Funding
- National Natural Science Foundation of China [32171465, 82102392]
- National Science and Technology Major Project of the Ministry of Science and Technology of China [2018ZX10301402]
- General Program of the Natural Science Foundation of Guangdong Province of China [2021A1515012438]
- National Postdoctoral Program for Innovative Talent [BX20200398]
- China Postdoctoral Science Foundation [2020M672995]
- Guangdong Basic and Applied Basic Research Foundation [2020A1515110170]
- Characteristic Innovation Research Project of University Teachers [2020SWYY07]
- National Ten Thousand Plan-Young Top Talents of China
- Hubei Provincial Health Commission [WJ2019H312]
- major projects of Wuhan Municipal Health Commission [WX19M02]
- Foundation of Health Commission of Hubei Province of China [WJ2019Q008]
- Dongguan Science and Technology of Social Development Program [202050715007221]
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The development of a gene knockout chain reaction (GKCR) method in this study improves the efficiency of gene editing for the treatment of HPV-related cervical cancer. Through the disruption of the HPV18 E6 and E7 genes using this method, the expression of P53/RB proteins is upregulated and the proliferation and motility of cancer cells are inhibited.
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.
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