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Lentivirus pre-packed with Cas9 protein for safer gene editing

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Abstract

The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an ‘all in one virus’ to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications.

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References

  1. Gersbach CA, Perez-Pinera P . Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine. Expert Opin Ther Targets 2014; 18: 835–839.

    CAS  Article  Google Scholar 

  2. Niu J, Zhang B, Chen H . Applications of TALENs and CRISPR/Cas9 in human cells and their potentials for gene therapy. Mol Biotechnol 2014; 56: 681–688.

    CAS  Article  Google Scholar 

  3. Zhang F, Wen Y, Guo X . CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet 2014; 23: R40–R46.

    CAS  Article  Google Scholar 

  4. Manjunath N, Yi G, Dang Y, Shankar P . Newer gene editing technologies toward HIV gene therapy. Viruses 2013; 5: 2748–2766.

    CAS  Article  Google Scholar 

  5. Riordan SM, Heruth DP, Zhang LQ, Ye SQ . Application of CRISPR/Cas9 for biomedical discoveries. Cell Biosci 2015; 5: 33.

    Article  Google Scholar 

  6. Sternberg SH, Doudna JA . Expanding the Biologist's Toolkit with CRISPR-Cas9. Mol Cell 2015; 58: 568–574.

    CAS  Article  Google Scholar 

  7. Mali P, Esvelt KM, Church GM . Cas9 as a versatile tool for engineering biology. Nat Methods 2013; 10: 957–963.

    CAS  Article  Google Scholar 

  8. Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res 2014; 42: 7473–7485.

    CAS  Article  Google Scholar 

  9. Cradick TJ, Fine EJ, Antico CJ, Bao G . CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res 2013; 41: 9584–9592.

    CAS  Article  Google Scholar 

  10. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 2013; 31: 822–826.

    CAS  Article  Google Scholar 

  11. Cannon RO 3rd, Leon MB, Watson RM, Rosing DR, Epstein SE . Chest pain and ‘normal’ coronary arteries–role of small coronary arteries. Am J Cardiol 1985; 55: 50B–60B.

    Article  Google Scholar 

  12. Wu X, Kriz AJ, Sharp PA . Target specificity of the CRISPR-Cas9 system. Quant Biol 2014; 2: 59–70.

    CAS  Article  Google Scholar 

  13. Skipper KA, Mikkelsen JG . Delivering the goods for genome engineering and editing. Hum Gene Ther 2015; 26: 486–497.

    CAS  Article  Google Scholar 

  14. Cai Y, Bak RO, Krogh LB, Staunstrup NH, Moldt B, Corydon TJ et al. DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors. Nucleic Acids Res 2014; 42: e28.

    CAS  Article  Google Scholar 

  15. Cai Y, Bak RO, Mikkelsen JG . Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases. Elife 2014; 3: e01911.

    Article  Google Scholar 

  16. Cai Y, Mikkelsen JG . Driving DNA transposition by lentiviral protein transduction. Mob Genet elements 2014; 4: e29591.

    Article  Google Scholar 

  17. Urano E, Aoki T, Futahashi Y, Murakami T, Morikawa Y, Yamamoto N et al. Substitution of the myristoylation signal of human immunodeficiency virus type 1 Pr55Gag with the phospholipase C-delta1 pleckstrin homology domain results in infectious pseudovirion production. J Gen Virol 2008; 89: 3144–3149.

    CAS  Article  Google Scholar 

  18. Hu W, Kaminski R, Yang F, Zhang Y, Cosentino L, Li F et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc Natl Acad Sci USA 2014; 111: 11461–11466.

    CAS  Article  Google Scholar 

  19. Ebina H, Misawa N, Kanemura Y, Koyanagi Y . Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep 2013; 3: 2510.

    Article  Google Scholar 

  20. Qu X, Wang P, Ding D, Li L, Wang H, Ma L et al. Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 proviral DNA from infected and latently infected human T cells. Nucleic Acids Res 2013; 41: 7771–7782.

    CAS  Article  Google Scholar 

  21. Swanson CM, Malim MH . SnapShot: HIV-1 proteins. Cell 2008; 133: 742, 742 e1.

    Article  Google Scholar 

  22. Yi G, Choi JG, Bharaj P, Abraham S, Dang Y, Kafri T et al. CCR5 gene editing of resting CD4(+) T cells by transient ZFN expression from HIV envelope pseudotyped nonintegrating lentivirus confers HIV-1 resistance in humanized mice. Mol Ther Nucleic Acids 2014; 3: e198.

    CAS  Article  Google Scholar 

  23. Klase Z, Yedavalli VS, Houzet L, Perkins M, Maldarelli F, Brenchley J et al. Activation of HIV-1 from latent infection via synergy of RUNX1 inhibitor Ro5-3335 and SAHA. PLoS Pathog 2014; 10: e1003997.

    Article  Google Scholar 

  24. al Yacoub N, Romanowska M, Haritonova N, Foerster J . Optimized production and concentration of lentiviral vectors containing large inserts. J Gene Med 2007; 9: 579–584.

    CAS  Article  Google Scholar 

  25. Lee SK, Dykxhoorn DM, Kumar P, Ranjbar S, Song E, Maliszewski LE et al. Lentiviral delivery of short hairpin RNAs protects CD4 T cells from multiple clades and primary isolates of HIV. Blood 2005; 106: 818–826.

    CAS  Article  Google Scholar 

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Correspondence to N Manjunath.

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Choi, J., Dang, Y., Abraham, S. et al. Lentivirus pre-packed with Cas9 protein for safer gene editing. Gene Ther 23, 627–633 (2016). https://doi.org/10.1038/gt.2016.27

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