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. 2021 Dec 18:24:171-179.
doi: 10.1016/j.omto.2021.12.011. eCollection 2022 Mar 17.

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

Rui Tian  1 , Jiashuo Liu  2 , Weiwen Fan  2 , Rui Li  3 , Zifeng Cui  2 , Zhuang Jin  2 , Zhaoyue Huang  2 , Hongxian Xie  4 , Lifang Li  2 , Zheying Huang  2 , Zheng Hu  2   5 , Ping Zhou  6 , Xun Tian  3
Affiliations

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

Rui Tian et al. Mol Ther Oncolytics. .

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.

Keywords: CRISPR-Cas9; HITI; HPV; cervical cancer; gene knockout chain reaction (GKCR).

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

None
Graphical abstract
Figure 1
Figure 1
Schematic outlining the GKCR
Figure 2
Figure 2
The Cas9/gRNA cassette could be integrated into the AAVS1 locus by the GKCR method in HEK293T cells (A) Schematic of the GKCR methods at the AAVS1 locus. (B and C) Agarose gel electrophoresis (B) and Sanger sequencing (C) of PCR products to verify Cas9/gRNA cassette integration into the targeted AAVS1 locus. (D) PCR and Sanger sequencing verified that the inserted Cas9/gRNA cassette was fully retained.
Figure 3
Figure 3
GKCR method significantly knocked out AAVS1 alleles in HEK293T cells (A) Schematic of the possible editing results of the two AAVS1 alleles in one HEK293T cell. (B) Editing results of two monoclonals in the AAVS1-GKCR group, verified by agarose gel electrophoresis and Sanger sequencing. (C) TIDE assays revealed that the GKCR method induced a significantly higher indel percentage than the control method at the AAVS1 locus. Data are presented as means ± SD (n = 3 per group). ∗∗p < 0.01; n.s., not significant; analyzed by one-way ANOVA and Tukey’s post hoc test.
Figure 4
Figure 4
GKCR method highly knocked out HPV18 E6 and E7 oncogenes in the HeLa cells (A and B) Agarose gel electrophoresis (A) and Sanger sequencing (B) of PCR products to verify that the Cas9/gRNA cassette integrated into the targeted HPV18 E6 and E7 locus. (C) Fold change of forward and reverse integration of the corresponding Cas9/gRNA cassettes into the respective target sites, analyzed by qPCR. Data are presented as mean ± SD (n = 3 per group). ∗∗p < 0.01, ∗∗∗p < 0.001; analyzed by two-tailed Students’ t test. (D) TIDE assays revealed that the GKCR method induced a significantly higher indel percentage than the control method at the HPV18 E6 and E7 locus. Data are presented as mean ± SD (n = 3 per group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; analyzed by one-way ANOVA and Tukey’s post hoc test.
Figure 5
Figure 5
GKCR method upregulated the P53/RB proteins and inhibited the proliferation and motility of the HeLa cells by targeting the HPV18 E6/E7 oncogenes (A) Western blot analysis. ∗p < 0.05, ∗∗p < 0.01; analyzed by two-tailed Students’ t test. (B) CCK-8 assay. ∗p < 0.05, analyzed by two-way RM ANOVA with Sidak’s multiple comparisons test. (C) Wound healing assay; scale bars, 250 μm. ∗p < 0.05, ∗∗p < 0.01; analyzed by two-tailed Students’ t test.
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