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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
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.
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.
Zhang F, Wen Y, Guo X . CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet 2014; 23: R40–R46.
Manjunath N, Yi G, Dang Y, Shankar P . Newer gene editing technologies toward HIV gene therapy. Viruses 2013; 5: 2748–2766.
Riordan SM, Heruth DP, Zhang LQ, Ye SQ . Application of CRISPR/Cas9 for biomedical discoveries. Cell Biosci 2015; 5: 33.
Sternberg SH, Doudna JA . Expanding the Biologist's Toolkit with CRISPR-Cas9. Mol Cell 2015; 58: 568–574.
Mali P, Esvelt KM, Church GM . Cas9 as a versatile tool for engineering biology. Nat Methods 2013; 10: 957–963.
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.
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.
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.
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.
Wu X, Kriz AJ, Sharp PA . Target specificity of the CRISPR-Cas9 system. Quant Biol 2014; 2: 59–70.
Skipper KA, Mikkelsen JG . Delivering the goods for genome engineering and editing. Hum Gene Ther 2015; 26: 486–497.
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.
Cai Y, Bak RO, Mikkelsen JG . Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases. Elife 2014; 3: e01911.
Cai Y, Mikkelsen JG . Driving DNA transposition by lentiviral protein transduction. Mob Genet elements 2014; 4: e29591.
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.
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.
Ebina H, Misawa N, Kanemura Y, Koyanagi Y . Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep 2013; 3: 2510.
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.
Swanson CM, Malim MH . SnapShot: HIV-1 proteins. Cell 2008; 133: 742, 742 e1.
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.
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.
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.
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.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Supplementary Information accompanies this paper on Gene Therapy website
Supplementary information
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/gt.2016.27
This article is cited by
-
Viral vectors and extracellular vesicles: innate delivery systems utilized in CRISPR/Cas-mediated cancer therapy
Cancer Gene Therapy (2023)
-
Maybe you can turn me on: CRISPRa-based strategies for therapeutic applications
Cellular and Molecular Life Sciences (2022)
-
Prevalence of CCR5delta32 in Northeastern Iran
BMC Medical Genetics (2019)
-
Novel gRNA design pipeline to develop broad-spectrum CRISPR/Cas9 gRNAs for safe targeting of the HIV-1 quasispecies in patients
Scientific Reports (2019)
-
The therapeutic landscape of HIV-1 via genome editing
AIDS Research and Therapy (2017)