CRISPR-Cas9 Delivery by Artificial Virus (RRPHC)

  • Suleixin Yang
  • Qinjie Wu
  • Yuquan Wei
  • Changyang Gong
Part of the Methods in Molecular Biology book series (MIMB, volume 1961)


Since its first harnessing in gene editing in 2012 and successful application in mammalian gene editing in 2013, the CRISPR-Cas9 system exerted magnificent power in all gene-editing-related applications, indicating a sharp thrive of this novel technology. However, there are still some critical drawbacks of the CRISPR-Cas9 system that hampered its broad application in gene editing. Efficient delivery of the Cas9 protein and its partner small guide RNA (sgRNA) to the target cells or tissue is one of the technical bottlenecks. CRISPR-Cas9 delivery via DNA plasmids still plays the big role in gene editing methods. With regard to the disadvantages of CRISPR-Cas9 plasmids, the most acute barrier lies in its large size (>10 kb) and the subsequent low transfection efficiency by conventional transfection method. In this chapter, what we present is an easy method by fabricating CRISPR-Cas9 plasmids into nanoparticle system and efficiently delivered into target cells to achieve gene editing.

Key words

CRISPR-Cas9 Transfection Artificial virus Branched polyethylene imine Heptafluorobutyric anhydride 


  1. 1.
    Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308. Scholar
  2. 2.
    Wang HY, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918. Scholar
  3. 3.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278. Scholar
  4. 4.
    Yang L, Mali P, Kim-Kiselak C, Church G (2014) CRISPR-Cas-mediated targeted genome editing in human cells. Methods Mol Biol 1114:245–267. Scholar
  5. 5.
    Weeks DP, Jiang WZ (2014) Genome editing in plants using CRISPR/Cas9/sgRNA technologies. In Vitro Cell Dev-An 50:S21–S21Google Scholar
  6. 6.
    Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14:327. Scholar
  7. 7.
    Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V (2015) Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol 32:76–84. Scholar
  8. 8.
    Butler M, van der Meer L, Yu J, Beeby T, Kuiper R, van Leeuwen F (2017) CRISPR/CAS9 based reverse genetics identifies therapeutic targets to improve therapy response in paediatric acute lymphoblastic leukemia. Pediatr Blood Cancer 64:S5–S6Google Scholar
  9. 9.
    Gonzalez F (2016) CRISPR/Cas9 genome editing in human pluripotent stem cells: harnessing human genetics in a dish. Dev Dyn 245(7):788–806. Scholar
  10. 10.
    Naylor J, Suckow AT, Seth A, Baker DJ, Sermadiras I, Ravn P, Howes R, Li JL, Snaith MR, Coghlan MP, Hornigold DC (2016) Use of CRISPR/Cas9-engineered INS-1 pancreatic beta cells to define the pharmacology of dual GIPR/GLP-1R agonists. Biochem J 473:2881–2891. Scholar
  11. 11.
    Cox DBT, Platt RJ, Zhang F (2015) Therapeutic genome editing: prospects and challenges. Nat Med 21(2):121–131CrossRefGoogle Scholar
  12. 12.
    Li L, He ZY, Wei XW, Gao GP, Wei YQ (2015) Challenges in CRISPR/CAS9 delivery: potential roles of nonviral vectors. Hum Gene Ther 26(7):452–462. Scholar
  13. 13.
    Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096. Scholar
  14. 14.
    Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F (2014) CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159(2):440–455. Scholar
  15. 15.
    Senis E, Fatouros C, Grosse S, Wiedtke E, Niopek D, Mueller AK, Borner K, Grimm D (2014) CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol J 9(11):1402–1412. Scholar
  16. 16.
    Kennedy EM, Cullen BR (2015) Bacterial CRISPR/Cas DNA endonucleases: a revolutionary technology that could dramatically impact viral research and treatment. Virology 479–480:213–220. Scholar
  17. 17.
    Cheong TC, Compagno M, Chiarle R (2016) Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system. Nat Commun 7:10934. Scholar
  18. 18.
    Fricano-Kugler CJ, Williams MR, Salinaro JR, Li M, Luikart B (2016) Designing, packaging, and delivery of high titer CRISPR retro and lentiviruses via stereotaxic injection. J Vis Exp 111.
  19. 19.
    Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG (2014) Non-viral vectors for gene-based therapy. Nat Rev Genet 15(8):541–555. Scholar
  20. 20.
    Li L, Song LJ, Liu XW, Yang X, Li X, He T, Wang N, Yang SLX, Yu C, Yin T, Wen YZ, He ZY, Wei XW, Su WJ, Wu QJ, Yao SH, Gong CY, Wei YQ (2017) Artificial virus delivers CRISPR-Cas9 system for genome editing of cells in mice. ACS Nano 11(1):95–111. Scholar
  21. 21.
    Li L, Song LJ, Yang X, Li X, Wu YZ, He T, Wang N, Yang SLX, Zeng Y, Yang L, Wu QJ, Wei YQ, Gong CY (2016) Multifunctional “core-shell” nanoparticles-based gene delivery for treatment of aggressive melanoma. Biomaterials 111:124–137. Scholar
  22. 22.
    Horváth IT, Rábai J (1994) Facile catalyst separation without water: fluorous biphase hydroformylation of olefins. Science 266(5182):72CrossRefGoogle Scholar
  23. 23.
    Wang MM, Liu HM, Li L, Cheng YY (2014) A fluorinated dendrimer achieves excellent gene transfection efficacy at extremely low nitrogen to phosphorus ratios. Nat Commun 5:3053. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Suleixin Yang
    • 1
  • Qinjie Wu
    • 1
  • Yuquan Wei
    • 1
  • Changyang Gong
    • 1
  1. 1.State Key Laboratory of Biotherapy and Cancer Center, West China HospitalSichuan UniversityChengduChina

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