Light-responsive charge-reversal nanovector for high-efficiency in vivo CRISPR/Cas9 gene editing with controllable location and time


Controllably and efficaciously localized CRISPR/Cas9 plasmids transfection plays an essential role in genetic editing associated with various key human diseases. We employed near-infrared (NIR) light-responsive CRISPR/Cas9 plasmids delivery via a charge-reversal nanovector to achieve highly efficient and site-specific gene editing. The nanovector with abundant positive charges was fabricated on the basis of an ultraviolet-sensitive conjugated polyelectrolyte coated on an upconversion nanomaterial (UCNP-UVP-P), which can convert into negative charges upon 980 nm light irradiation. Using the as-prepared nanovector, we demonstrated the plasmids could be efficiently transfected into tumor cells (∼ 63% ± 4%) in a time-controlled manner, and that functional CRISPR/Cas9 proteins could be successfully expressed in a selected NIR-irradiated region. Particularly, this strategy was successfully applied to the delivery of CRISPR/Cas9 gene to tumor cells in vivo, inducing high efficiency editing of the target gene PLK-1 under photoirradiation. Therefore, this precisely controlled gene regulation strategy has the potential to serve as a new paradigm for gene engineering in complex biological systems.

This is a preview of subscription content, log in to check access.


  1. [1]

    Barrangou, R.; Doudna, J. A. Applications of CRISPR technologies in research and beyond. Nat. Biotechnol.2016, 34, 933–941.

    CAS  Article  Google Scholar 

  2. [2]

    Long, C. Z.; McAnally, J. R.; Shelton, J. M.; Mireault, A. A.; Bassel-Duby, R.; Olson, E. N. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science2014, 345, 1184–1188.

    CAS  Article  Google Scholar 

  3. [3]

    Wang, H. X.; Li, M. Q.; Lee, C. M.; Chakraborty, S.; Kim, H. W.; Bao, G; Leong, K. W. CRISPR/Cas9-based genome editing for disease modeling and therapy: Challenges and opportunities for nonviral delivery. Chem. Rev.2017, 117, 9874–9906.

    CAS  Article  Google Scholar 

  4. [4]

    Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science2012, 337, 816–821.

    CAS  Article  Google Scholar 

  5. [5]

    Sánchez-Rivera, F. J.; Jacks, T. Applications of the CRISPR-Cas9 system in cancer biology. Nat. Rev. Cancer2015, 15, 387–395.

    Article  CAS  Google Scholar 

  6. [6]

    Swiech, L.; Heidenreich, M.; Banerjee, A.; Habib, N.; Li, Y. Q.; Trombetta, J.; Sur, M.; Zhang, F. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol.2015, 33, 102–106.

    CAS  Article  Google Scholar 

  7. [7]

    Hsu, P. D.; Lander, E. S.; Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell2014, 157, 1262–1278.

    CAS  Article  Google Scholar 

  8. [8]

    Kiani, S.; Chavez, A.; Tuttle, M.; Hall, R. N.; Chari, R.; Ter-Ovanesyan, D.; Qian, J.; Pruitt, B. W.; Beal, J.; Vora, S. et al. Cas9 gRNA engineering for genome editing, activation and repression. Nat. Methods2015, 12, 1051–1054.

    CAS  Article  Google Scholar 

  9. [9]

    Platt, R. J.; Chen, S. D.; Zhou, Y.; Yim, M. J.; Swiech, L.; Kempton, H. R.; Dahlman, J. E.; Parnas, O.; Eisenhaure, T. M.; Jovanovic, M. et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell2014, 159, 440–455.

    CAS  Article  Google Scholar 

  10. [10]

    Xue, W.; Chen, S. D.; Yin, H.; Tammela, T.; Papagiannakopoulos, T.; Joshi, N. S.; Cai, W. X.; Yang, G.; Bronson, R.; Crowley, D. G. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature2014, 514, 380–384.

    CAS  Article  Google Scholar 

  11. [11]

    Liang, X. Q.; Potter, J.; Kumar, S.; Zou, Y. F.; Quintanilla, R.; Sridharan, M.; Carte, J.; Chen, W.; Roark, N.; Ranganathan, S. et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J. Biotechnol.2015, 208, 44–53.

    CAS  Article  Google Scholar 

  12. [12]

    Horwitz, A. A.; Walter, J. M.; Schubert, M. G.; Kung, S. H.; Hawkins, K.; Platt, D. M.; Hernday, A. D.; Mahatdejkul-Meadows, T.; Szeto, W.; Chandran, S. S. et al. Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR-Cas. Cell Syst.2015, 1, 88–96.

    CAS  Article  Google Scholar 

  13. [13]

    Svitashev, S.; Young, J. K.; Schwartz, C.; Gao, H. R.; Falco, S. C.; Cigan, A. M. Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol.2015, 169, 931–945.

    Article  CAS  Google Scholar 

  14. [14]

    Chu, V. T.; Weber, T.; Wefers, B.; Wurst, W.; Sander, S.; Rajewsky, K.; Kühn, R. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol.2015, 33, 543–548.

    CAS  Article  Google Scholar 

  15. [15]

    Garneau, J. E.; Dupuis, M. È.; Villion, M.; Romero, D. A.; Barrangou, R.; Boyaval, P.; Fremaux, C.; Horvath, P.; Magadan, A. H.; Moineau, S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature2010, 468, 67–71.

    CAS  Article  Google Scholar 

  16. [16]

    Liu, Q.; Zhao, K.; Wang, C.; Zhang, Z. Z.; Zheng, C. X.; Zhao, Y.; Zheng, Y. D.; Liu, C. Y.; An, Y. L.; Shi, L. Q. et al. Multistage delivery nanoparticle facilitates efficient CRISPR/dCas9 activation and tumor growth suppression in vivo. Adv. Sci.2019, 6, 1801423.

    Article  CAS  Google Scholar 

  17. [17]

    Wang, P.; Zhang, L. M.; Zheng, W. F.; Cong, L. M.; Guo, Z. R.; Xie, Y. Z. Y.; Wang, L.; Tang, R. B.; Feng, Q.; Hamada, Y. et al. Thermo-triggered release of CRISPR-Cas9 system by lipid — encapsulated gold nanoparticles for tumor therapy. Angew. Chem., Int. Ed.2018, 57, 1491–1496.

    CAS  Article  Google Scholar 

  18. [18]

    Yin, H.; Song, C. Q.; Dorkin, J. R.; Zhu, L. J.; Li, Y. X.; Wu, Q. Q.; Park, A.; Yang, J.; Suresh, S.; Bizhanova, A. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol.2016, 34, 328–333.

    CAS  Article  Google Scholar 

  19. [19]

    He, Z. Y.; Men, K.; Qin, Z.; Yang, Y.; Xu, T.; Wei, Y. Q. Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field. Sci. China Life Sci.2017, 60, 458–467.

    CAS  Article  Google Scholar 

  20. [20]

    Luten, J.; Van Nostrum, C. F.; De Smedt, S. C.; Hennink, W. E. Biodegradable polymers as non-viral carriers for plasmid DNA delivery. J. Controlled Release2008, 126, 97–110.

    CAS  Article  Google Scholar 

  21. [21]

    Wang, H. X.; Song, Z. Y.; Lao, Y. H.; Xu, X.; Gong, J.; Cheng, D.; Chakraborty, S.; Park, J. S.; Li, M. Q.; Huang, D. T. et al. Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide. Proc. Natl. Acad. Sci. USA2018, 115, 4903–4908.

    CAS  Article  Google Scholar 

  22. [22]

    Wang, P.; Zhang, L. M.; Xie, Y. Z. Y.; Wang, N. X.; Tang, R. B.; Zheng, W. F.; Jiang, X. Y. Genome editing for cancer therapy: Delivery of Cas9 protein/sgRNA plasmid via a gold nanocluster/lipid core-shell nanocarrier. Adv. Sci.2017, 4, 1700175.

    Article  CAS  Google Scholar 

  23. [23]

    Yu, X.; Liang, X. Q.; Xie, H. M.; Kumar, S.; Ravinder, N.; Potter, J.; du Jeu, X. D. M.; Chesnut, J. D. Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX. Biotechnol. Lett.2016, 38, 919–929.

    CAS  Article  Google Scholar 

  24. [24]

    Zhang, L. M.; Wang, P.; Feng, Q.; Wang, N. X.; Chen, Z. T.; Huang, Y. Y.; Zheng, W. F.; Jiang, X. Y. Lipid nanoparticle-mediated efficient delivery of CRISPR/Cas9 for tumor therapy. NPG Asia Materi.2017, 9, e441.

    CAS  Article  Google Scholar 

  25. [25]

    Li, L.; Hu, S.; Chen, X. Y. Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities. Biomaterials2018, 171, 207–218.

    CAS  Article  Google Scholar 

  26. [26]

    Li, L.; He, Z. Y.; Wei, X. W.; Gao, G. P.; Wei, Y. Q. Challenges in CRISPR/CAS9 delivery: Potential roles of nonviral vectors. Hum. Gene Ther.2015, 26, 452–462.

    CAS  Article  Google Scholar 

  27. [27]

    Sun, W. J.; Ji, W. Y.; Hall, J. M.; Hu, Q. Y.; Wang, C.; Beisel, C. L.; Gu, Z. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew. Chem, Int. Ed.2015, 54, 12029–12033.

    CAS  Article  Google Scholar 

  28. [28]

    Wang, L. Y.; Li, F. F.; Dang, L.; Liang, C.; Wang, C.; He, B.; Liu, J.; Li, D. F.; Wu, X. H.; Xu, X. G. et al. In vivo delivery systems for therapeutic genome editing. Int. J. Mol. Sci.2016, 17, 626.

    Article  CAS  Google Scholar 

  29. [29]

    Yan, H. J.; Oommen, O. P.; Yu, D.; Hilborn, J.; Qian, H.; Varghese, O. P. Chondroitin sulfate-coated DNA-nanoplexes enhance transfection efficiency by controlling plasmid release from endosomes: A new insight into modulating nonviral gene transfection. Adv. Funct. Mater.2015, 25, 3907–3915.

    CAS  Article  Google Scholar 

  30. [30]

    Guo, X.; Huang, L. Recent advances in nonviral vectors for gene delivery. Acc. Chem. Res.2012, 45, 971–979.

    CAS  Article  Google Scholar 

  31. [31]

    Zhou, M. X.; Huang, H.; Wang, D. Q.; Lu, H. R.; Chen, J.; Chai, Z. F.; Yao, S. Q.; Hu, Y. Light-triggered PEGylation/dePEGylation of the nanocarriers for enhanced tumor penetration. Nano Lett.2019, 19, 3671–3675.

    CAS  Article  Google Scholar 

  32. [32]

    Zhao, H.; Hu, W. B.; Ma, H. H.; Jiang, R. C.; Tang, Y. F.; Ji, Y.; Lu, X. M.; Hou, B.; Deng, W. X.; Huang, W. et al. Photo-induced chargevariable conjugated polyelectrolyte brushes encapsulating upconversion nanoparticles for promoted siRNA release and collaborative photodynamic therapy under NIR light irradiation. Adv. Funct. Mater.2017, 27, 1702592.

    Article  CAS  Google Scholar 

  33. [33]

    Cong, Y.; Ji, L.; Gao, Y. J.; Liu, F. H.; Cheng, D. B.; Hu, Z. Y.; Qiao, Z. Y.; Wang, H. Microenvironment-induced in situ self-assembly of polymer–peptide conjugates that attack solid tumors deeply. Angew. Chem., Int. Ed.2019, 58, 4632–4637.

    CAS  Article  Google Scholar 

  34. [34]

    Pan, Y. C.; Yang, J. J.; Luan, X. W.; Liu, X. L.; Li, X. Q.; Yang, J.; Huang, T.; Sun, L.; Wang, Y. Z.; Lin, Y. H. et al. Near-infrared upconversion–activated CRISPR-Cas9 system: A remote-controlled gene editing platform. Sci. Adv.2019, 5, eaav7199.

    CAS  Article  Google Scholar 

  35. [35]

    Xiong, Q. R.; Lim, Y.; Li, D.; Pu, K. Y.; Liang, L.; Duan, H. W. Photoactive nanocarriers for controlled delivery. Adv. Funct. Mater.2020, 30, 1903896.

    CAS  Article  Google Scholar 

  36. [36]

    Pansare, V. J.; Hejazi, S.; Faenza, W. J.; Prud’homme, R. K. Review of long-wavelength optical and NIR imaging materials: Contrast agents, fluorophores, and multifunctional nano carriers. Chem. Mater.2012, 24, 812–827.

    CAS  Article  Google Scholar 

  37. [37]

    Wang, C.; Cheng, L.; Liu, Y. M.; Wang, X. J.; Ma, X. X.; Deng, Z. Y.; Li, Y. G.; Liu, Z. Imaging-guided pH-sensitive photodynamic therapy using charge reversible upconversion nanoparticles under near-infrared light. Adv. Funct. Mater.2013, 23, 3077–3086.

    CAS  Article  Google Scholar 

  38. [38]

    Zhao, J.; Chu, H. Q.; Zhao, Y.; Lu, Y.; Li, L. L. A NIR light gated DNA nanodevice for spatiotemporally controlled imaging of MicroRNA in cells and animals. J. Am. Chem. Soc.2019, 141, 7056–7062.

    CAS  Article  Google Scholar 

  39. [39]

    Li, L.; Yang, Z.; Zhu, S. J.; He, L. C.; Fan, W. P.; Tang, W.; Zou, J. H.; Shen, Z. Y.; Zhang, M. R.; Tang, L. G. et al. A rationally designed semiconducting polymer brush for NIR-II imaging-guided light-triggered remote control of CRISPR/Cas9 genome editing. Adv. Mater.2019, 31, 1901187.

    Article  CAS  Google Scholar 

  40. [40]

    Lyu, Y.; He, S. S.; Li, J. C.; Jiang, Y. Y.; Sun, H.; Miao, Y. S.; Pu, K. Y. A photolabile semiconducting polymer nanotransducer for near-infrared regulation of CRISPR/Cas9 gene editing. Angew. Chem., Int. Ed.2019, 58, 18197–18201.

    CAS  Article  Google Scholar 

  41. [41]

    Dobson, J. Gene therapy progress and prospects: Magnetic nanoparticle-based gene delivery. Gene Ther.2006, 13, 283–287.

    CAS  Article  Google Scholar 

  42. [42]

    Zhu, H. B.; Zhang, L. L.; Tong, S.; Lee, C. M.; Deshmukh, H.; Bao, G. Spatial control of in vivo CRISPR–Cas9 genome editing via nanomagnets. Nat. Biomed. Eng.2019, 3, 126–136.

    CAS  Article  Google Scholar 

  43. [43]

    Chen, G. Y.; Qiu, H. L.; Prasad, P. N.; Chen, X. Y. Upconversion nanoparticles: Design, nanochemistry, and applications in theranostics. Chem. Rev.2014, 114, 5161–5214.

    CAS  Article  Google Scholar 

  44. [44]

    Chinen, A. B.; Guan, C. M.; Ferrer, J. R.; Barnaby, S. N.; Merkel, T. J.; Mirkin, C. A. Nanoparticle probes for the detection of cancer bio-markers, cells, and tissues by fluorescence. Chem. Rev.2015, 115, 10530–10574.

    CAS  Article  Google Scholar 

  45. [45]

    Liu, Y.; Chen, M.; Cao, T. Y.; Sun, Y.; Li, C. Y.; Liu, Q.; Yang, T. S.; Yao, L. M.; Feng, W.; Li, F. Y. A cyanine-modified nanosystem for in vivo upconversion luminescence bioimaging of methylmercury. J. Am. Chem. Soc.2013, 135, 9869–9876.

    CAS  Article  Google Scholar 

  46. [46]

    Li, Z. H.; Yuan, H.; Yuan, W.; Su, Q. Q.; Li, F. Y. Upconversion nano-probes for biodetections. Coordin. Chem. Rev.2018, 354, 155–168.

    CAS  Article  Google Scholar 

  47. [47]

    Wang, M.; Mi, C. C.; Wang, W. X.; Liu, C. H.; Wu, Y. F.; Xu, Z. R.; Mao, C. B.; Xu, S. K. Immunolabeling and NIR-excited fluorescent imaging of HeLa cells by using NaYF4:Yb,Er upconversion nanoparticles. ACS Nano2009, 3, 1580–1586.

    CAS  Article  Google Scholar 

  48. [48]

    Vetrone, F.; Naccache, R.; Zamarrón, A.; Juarranz De La Fuente, A.; Sanz-Rodriguez, F.; Martinez Maestro, L.; Martin Rodriguez, E.; Jaque, D.; Garcia Solé, J.; Capobianco, J. A. Temperature sensing using fluorescent nanothermometers. ACS Nano2010, 4, 3254–3258.

    CAS  Article  Google Scholar 

  49. [49]

    Zheng, J. D.; Wu, Y. X.; Xing, D.; Zhang, T. Synchronous detection of glutathione/hydrogen peroxide for monitoring redox status in vivo with a ratiometric upconverting nanoprobe. Nano Res.2019, 12, 931–938.

    CAS  Article  Google Scholar 

  50. [50]

    Yang, Y. M.; Shao, Q.; Deng, R. R.; Wang, C.; Teng, X.; Cheng, K.; Cheng, Z.; Huang, L.; Liu, Z.; Liu, X. G. et al. In vitro and in vivo uncaging and bioluminescence imaging by using photocaged upcon-version nanoparticles. Angew. Chem., Int. Ed.2012, 51, 3125–3129.

    CAS  Article  Google Scholar 

  51. [51]

    Yang, D. M.; Ma, P. A.; Hou, Z. Y.; Cheng, Z. Y.; Li, C. X.; Lin, J. Current advances in lanthanide ion (Ln3+)-based upconversion nano-materials for drug delivery. Chem. Soc. Rev.2015, 44, 1416–1448.

    CAS  Article  Google Scholar 

  52. [52]

    Peng, J. J.; Sun, Y.; Liu, Q.; Yang, Y.; Zhou, J.; Feng, W.; Zhang, X. Z.; Li, F. Y. Upconversion nanoparticles dramatically promote plant growth without toxicity. Nano Res.2012, 5, 770–782.

    CAS  Article  Google Scholar 

  53. [53]

    Kang, H.; Rho, S.; Stiles, W. R.; Hu, S.; Baek, Y.; Hwang, D. W.; Kashiwagi, S.; Moon S. K.; Choi, H. S. Size-dependent EPR effect of polymeric nanoparticles on tumor targeting. Adv. Healthcare Mater.2020, 9, 1901223.

    CAS  Article  Google Scholar 

  54. [54]

    Mali, P.; Yang, L. H.; Esvelt, K. M.; Aach, J.; Guell, M.; DiCarlo, J. E.; Norville, J. E.; Church, G. M. RNA-guided human genome engineering via Cas9. Science2013, 339, 823–826.

    CAS  Article  Google Scholar 

  55. [55]

    Ran, F. A.; Hsu, P. D.; Lin, C. Y.; Gootenberg, J. S.; Konermann, S.; Trevino, A. E.; Scott, D. A.; Inoue, A.; Matoba, S.; Zhang, Y. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell2013, 154, 1380–1389.

    CAS  Article  Google Scholar 

  56. [56]

    Spänkuch-Schmitt, B.; Bereiter-Hahn, J.; Kaufmann, M.; Strebhardt, K. Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells. J. Natl. Cancer Inst.2002, 94, 1863–1877.

    Article  Google Scholar 

Download references


This research was supported by the National Natural Science Foundation of China (Nos. 21771065 and 81630046); the Guangdong Special Support Program (No. 2017TQ04R138), the Natural Science Foundation of Guangdong (No. 2019A1515012021), the Science and Technology Planning Project of Guangdong (No. 2017A020215088), Pearl River Nova Program of Guangzhou (No. 201806010189).

Author information



Corresponding authors

Correspondence to Tao Zhang or Da Xing.

Electronic Supplementary Material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wu, Y., Zheng, J., Zeng, Q. et al. Light-responsive charge-reversal nanovector for high-efficiency in vivo CRISPR/Cas9 gene editing with controllable location and time. Nano Res. (2020).

Download citation


  • light-responsive
  • charge-reversal
  • CRISPR/Cas9
  • gene editing