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Non-homologous dsODN increases the mutagenic effects of CRISPR-Cas9 to disrupt oncogene E7 in HPV positive cells

Abstract

Genome editing tools targeting high-risk human papillomavirus (HPV) oncogene could be a promising therapeutic strategy for the treatment of HPV-related cervical cancer. We aimed to improve the editing efficiency and detect off-target effects concurrently for the clinical translation strategy by using CRISPR-Cas9 system co-transfected with 34nt non-homologous double-stranded oligodeoxynucleotide (dsODN). We firstly tested this strategy on targeting the Green Fluorescent Protein (GFP) gene, of which the expression is easily observed. Our results showed that the GFP+ cells were significantly decreased when using GFP-sgRNAs with dsODN, compared to using GFP-sgRNAs without donors. By PCR and Sanger sequencing, we verified the dsODN integration into the break sites of the GFP gene. And by amplicon sequencing, we observed that the indels% of the targeted site on the GFP gene was increased by using GFP-sgRNAs with dsODN. Next, we went on to target the HPV18 E7 oncogene by using single E7-sgRNA and multiplexed E7-sgRNAs respectively. Whenever using single sgRNA or multiplexed sgRNAs, the mRNA expression of HPV18 E7 oncogene was significantly decreased when adding E7-sgRNAs with dsODN, compared to E7-sgRNAs without donor. And the indels% of the targeted sites on the HPV18 E7 gene was markedly increased by adding dsODN with E7-sgRNAs. Finally, we performed GUIDE-Seq to verify that the integrated dsODN could serve as the marker to detect off-target effects in using single or multiplexed two sgRNAs. And we detected fewer on-target reads and off-target sites in multiplexes compared to the single sgRNAs when targeting the GFP and the HPV18 E7 genes. Together, CRISPR-Cas9 system co-transfected with 34nt dsODN concurrently improved the editing efficiency and monitored off-target effects, which might provide new insights in the treatment of HPV infections and related cervical cancer.

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Fig. 1: CRISPR-Cas9 co-electrotransfection with non-homologous ODNs disrupted the GFP gene expression in GFP+ HeLa cells.
Fig. 2: Non-homologous dsODN was integrated into the DSB sites and promoted indels of the GFP gene in GFP+ HeLa cells.
Fig. 3: HPV18 E7-sgRNAs co-electrotransfected with non-homologous dsODN enhanced the disruption of the HPV18 E7 gene in HeLa cells.
Fig. 4: The disruption efficiency of the HPV18 E7 gene using multiplexed two sgRNAs co-electrotransfected with non-homologous dsODN in HeLa cells.
Fig. 5: Co-electrotransfected dsODN integration sites were analyzed to identify off-target effects in HeLa cells.

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Acknowledgements

This work was supported by the National Science and Technology Major Project of the Ministry of science and technology of China (No. 2018ZX10301402); National Ten Thousand Plan-Young Top Talents of China; General Program of Natural Science Foundation of Guangdong Province of China (No. 2021A1515012438); the National Postdoctoral Program for Innovative Talent (No. BX20200398); the China Postdoctoral Science Foundation (No. 2020M672995); Guangdong Basic and Applied Basic Research Foundation (No.2020A1515110170); Characteristic Innovation Research Project of University Teachers (No. 2020SWYY07).

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Conceptualization: WF, HX, ZH; Data curation: WF, MY; Formal Analysis: WF, WX, MY, XW; Funding acquisition: ZH; Investigation: BD, KS, IH, and PD; Resources: ZH; Validation: RT, ZJ, HX; Software: ZC; Visualization: XT, BL; Writing original draft: WF; Writing – review & editing: WF, HX, ZH, JT.

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Correspondence to Jinfeng Tan, Hongyan Xu or Zheng Hu.

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Fan, W., Yu, M., Wang, X. et al. Non-homologous dsODN increases the mutagenic effects of CRISPR-Cas9 to disrupt oncogene E7 in HPV positive cells. Cancer Gene Ther 29, 758–769 (2022). https://doi.org/10.1038/s41417-021-00355-z

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