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

Cancer Gene Therapy volume 29, pages 758–769 (2022)Cite this article

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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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References

  1. Cohen PA, Jhingran A, Oaknin A, Denny L. Cervical cancer. Lancet. 2019;393:169–82.

    Article  Google Scholar 

  2. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424.

    Article  Google Scholar 

  3. Crosbie EJ, Einstein MH, Franceschi S, Kitchener HC. Human papillomavirus and cervical cancer. Lancet. 2013;382:889–99.

    Article  Google Scholar 

  4. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N. Engl J Med. 2003;348:518–27.

    Article  Google Scholar 

  5. Hu Z, Zhu D, Wang W, Li W, Jia W, Zeng X, et al. Genome-wide profiling of HPV integration in cervical cancer identifies clustered genomic hot spots and a potential microhomology-mediated integration mechanism. Nat Genet. 2015;47:158–63.

    Article  CAS  Google Scholar 

  6. Adey A, Burton JN, Kitzman JO, Hiatt JB, Lewis AP, Martin BK, et al. The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line. Nature. 2013;500:207–11.

    Article  CAS  Google Scholar 

  7. Nakamura Y, Migita T, Hosoda F, Okada N, Gotoh M, Arai Y, et al. Kruppel-like factor 12 plays a significant role in poorly differentiated gastric cancer progression. Int J Cancer. 2009;125:1859–67.

    Article  CAS  Google Scholar 

  8. Dong Z, Yang L, Lai D. KLF5 strengthens drug resistance of ovarian cancer stem-like cells by regulating survivin expression. Cell Prolif. 2013;46:425–35.

    Article  CAS  Google Scholar 

  9. Narisawa-Saito M, Kiyono T. Basic mechanisms of high-risk human papillomavirus-induced carcinogenesis: roles of E6 and E7 proteins. Cancer Sci. 2007;98:1505–11.

    Article  CAS  Google Scholar 

  10. Halbert CL, Demers GW, Galloway DA. The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J Virol. 1991;65:473–8.

    Article  CAS  Google Scholar 

  11. Barbosa MS, Vass WC, Lowy DR, Schiller JT. In vitro biological activities of the E6 and E7 genes vary among human papillomaviruses of different oncogenic potential. J Virol. 1991;65:292–8.

    Article  CAS  Google Scholar 

  12. Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science. 2010;329:1355–8.

    Article  CAS  Google Scholar 

  13. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6.

    Article  CAS  Google Scholar 

  14. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–78.

    Article  CAS  Google Scholar 

  15. Zhen S, Hua L, Takahashi Y, Narita S, Liu YH, Li Y. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9. Biochem Biophys Res Commun. 2014;450:1422–6.

    Article  CAS  Google Scholar 

  16. Zhen S, Hua L, Liu YH, Gao LC, Fu J, Wan DY, et al. Harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated Cas9 system to disrupt the hepatitis B virus. Gene Ther. 2015;22:404–12.

    Article  CAS  Google Scholar 

  17. Ye L, Wang J, Beyer AI, Teque F, Cradick TJ, Qi Z, et al. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Delta32 mutation confers resistance to HIV infection. Proc Natl Acad Sci USA. 2014;111:9591–6.

    Article  CAS  Google Scholar 

  18. Hu Z, Yu L, Zhu D, Ding W, Wang X, Zhang C, et al. Disruption of HPV16-E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells. Biomed Res Int. 2014;2014:612823.

    PubMed  PubMed Central  Google Scholar 

  19. Yoshiba T, Saga Y, Urabe M, Uchibori R, Matsubara S, Fujiwara H, et al. CRISPR/Cas9-mediated cervical cancer treatment targeting human papillomavirus E6. Oncol Lett. 2019;17:2197–206.

    CAS  PubMed  Google Scholar 

  20. Ling K, Yang L, Yang N, Chen M, Wang Y, Liang S, et al. Gene Targeting of HPV18 E6 and E7 Synchronously by Nonviral Transfection of CRISPR/Cas9 System in Cervical Cancer. Hum Gene Ther. 2020;31:297–308.

    Article  CAS  Google Scholar 

  21. Zhen S, Lu JJ, Wang LJ, Sun XM, Zhang JQ, Li X, et al. In vitro and in vivo synergistic therapeutic effect of cisplatin with human papillomavirus16 E6/E7 CRISPR/Cas9 on cervical cancer cell line. Transl Oncol. 2016;9:498–504.

    Article  Google Scholar 

  22. Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33:187–97.

    Article  CAS  Google Scholar 

  23. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.

    Article  CAS  Google Scholar 

  24. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–i890.

    Article  Google Scholar 

  25. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25:1754–60.

    Article  CAS  Google Scholar 

  26. You Q, Zhong Z, Ren Q, Hassan F, Zhang Y, Zhang T. CRISPRMatch: an automatic calculation and visualization tool for high-throughput CRISPR genome-editing data analysis. Int J Biol Sci. 2018;14:858–62.

    Article  CAS  Google Scholar 

  27. Chakravarthy A, Henderson S, Thirdborough SM, Ottensmeier CH, Su X, Lechner M, et al. Human papillomavirus drives tumor development throughout the head and neck: improved prognosis is associated with an immune response largely restricted to the oropharynx. J Clin Oncol. 2016;34:4132–41.

    Article  CAS  Google Scholar 

  28. Mirabello L, Yeager M, Yu K, Clifford GM, Xiao Y, Zhu B, et al. HPV16 E7 genetic conservation is critical to carcinogenesis. Cell. 2017;170:1164–74. e1166.

    Article  CAS  Google Scholar 

  29. Ding W, Hu Z, Zhu D, Jiang X, Yu L, Wang X, et al. Zinc finger nucleases targeting the human papillomavirus E7 oncogene induce E7 disruption and a transformed phenotype in HPV16/18-positive cervical cancer cells. Clin Cancer Res. 2014;20:6495–503.

    Article  CAS  Google Scholar 

  30. Hu Z, Ding W, Zhu D, Yu L, Jiang X, Wang X, et al. TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy. J Clin Invest. 2015;125:425–36.

    Article  Google Scholar 

  31. Gao X, Jin Z, Tan X, Zhang C, Zou C, Zhang W, et al. Hyperbranched poly(beta-amino ester) based polyplex nanopaticles for delivery of CRISPR/Cas9 system and treatment of HPV infection associated cervical cancer. J Control Release. 2020;321:654–68.

    Article  CAS  Google Scholar 

  32. Ceccaldi R, Rondinelli B, D’Andrea AD. Repair pathway choices and consequences at the double-strand break. Trends Cell Biol. 2016;26:52–64.

    Article  CAS  Google Scholar 

  33. Jasin M, Haber JE. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair. DNA Repair. 2016;44:6–16.

    Article  CAS  Google Scholar 

  34. Pannunzio NR, Watanabe G, Lieber MR. Nonhomologous DNA end-joining for repair of DNA double-strand breaks. J Biol Chem. 2018;293:10512–23.

    Article  CAS  Google Scholar 

  35. Xie A, Kwok A, Scully R. Role of mammalian Mre11 in classical and alternative nonhomologous end joining. Nat Struct Mol Biol. 2009;16:814–8.

    Article  CAS  Google Scholar 

  36. Xie A, Hartlerode A, Stucki M, Odate S, Puget N, Kwok A, et al. Distinct roles of chromatin-associated proteins MDC1 and 53BP1 in mammalian double-strand break repair. Mol Cell. 2007;28:1045–57.

    Article  CAS  Google Scholar 

  37. Guirouilh-Barbat J, Huck S, Bertrand P, Pirzio L, Desmaze C, Sabatier L, et al. Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells. Mol Cell. 2004;14:611–23.

    Article  CAS  Google Scholar 

  38. Guirouilh-Barbat J, Rass E, Plo I, Bertrand P, Lopez BS. Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends. Proc Natl Acad Sci USA. 2007;104:20902–7.

    Article  CAS  Google Scholar 

  39. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016;529:490–5.

    Article  CAS  Google Scholar 

Download references

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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  1. These authors contributed equally: Weiwen Fan, Miao Yu, Xin Wang

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China

    Weiwen Fan, Miao Yu, Weiling Xie, Rui Tian, Zifeng Cui, Zhuang Jin, Zhaoyue Huang, Jinfeng Tan & Zheng Hu

  2. Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

    Xin Wang

  3. Amity Institute of Molecular Medicine & Stem Cell Research, Amity University, Uttar Pradesh, Noida, India

    Bhudev C. Das

  4. Skolkovo Institute of Science and Technology, Moscow, Russia

    Konstantin Severinov

  5. Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Cape Town, South Africa

    Inga Isabel Hitzeroth

  6. Department of Zoology, North Orissa University, Bhubaneswar, Orissa, India

    Priya Ranjan Debata

  7. Department of Obstetrics and Gynecology, The Central Hospital of Wuhan, Wuhan, Hubei, China

    Xun Tian

  8. STech Company Bio-X Lab, Zhuhai, Guangdong, China

    Hongxian Xie

  9. School of Health Sciences and Sports, Macao Polytechnic Institute, Macao, China

    Bin Lang

  10. Department of Obstetrics and Gynecology, Yuebei People’s Hospital, Medical College of Shantou University, Guangzhou, Guangdong, China

    Hongyan Xu

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Contributions

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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