Article | Published:

Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations


Ex vivo CRISPR gene editing in haematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation, sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci, cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. The engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to those of non-treated cells, with no differences in differentiation. Here we demonstrate non-toxic delivery of the entire CRISPR payload into primary human blood progenitors.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Sequence data are available for download through the National Center for Biotechnology Information (BioProject ID: PRJNA529681).

Code availability

The bioinformatics pipeline used to analyse sequencing reads is available via GitHub (

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Hacein-Bey-Abina, S. et al. A modified gamma-retrovirus vector for X-linked severe combined immunodeficiency. N. Engl. J. Med. 371, 1407–1417 (2014).

  2. 2.

    Cicalese, M. P. et al. Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency. Blood 128, 45–54 (2016).

  3. 3.

    Sessa, M. et al. Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial. Lancet 388, 476–487 (2016).

  4. 4.

    Hacein-Bey Abina, S. et al. Outcomes following gene therapy in patients with severe Wiskott–Aldrich syndrome. J. Am. Med. Assoc. 313, 1550–1563 (2015).

  5. 5.

    Dunbar, C. E. et al. Gene therapy comes of age. Science 359, eaan4672 (2018).

  6. 6.

    Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419 (2003).

  7. 7.

    Hacein-Bey-Abina, S. et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348, 255–256 (2003).

  8. 8.

    Ott, M. G. et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat. Med. 12, 401–409 (2006).

  9. 9.

    Stein, S. et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat. Med. 16, 198–204 (2010).

  10. 10.

    Cornu, T. I., Mussolino, C. & Cathomen, T. Refining strategies to translate genome editing to the clinic. Nat. Med. 23, 415–423 (2017).

  11. 11.

    Chang, H. H. Y., Pannunzio, N. R., Adachi, N. & Lieber, M. R. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat. Rev. Mol. Cell Biol. 18, 495–506 (2017).

  12. 12.

    Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

  13. 13.

    Dever, D. P. et al. CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016).

  14. 14.

    De Ravin, S. S. et al. CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease. Sci. Transl. Med. 9, eaah3480 (2017).

  15. 15.

    Lefesvre, P., Attema, J. & van Bekkum, D. A comparison of efficacy and toxicity between electroporation and adenoviral gene transfer. BMC Mol. Biol. 3, 12 (2002).

  16. 16.

    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. 26, 452–462 (2015).

  17. 17.

    Finn, J. D. et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 22, 2227–2235 (2018).

  18. 18.

    Lee, K. et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat. Biomed. Eng. 1, 889–901 (2017).

  19. 19.

    Lee, B. et al. Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat. Biomed. Eng. 2, 497–507 (2018).

  20. 20.

    Rosi, N. L. et al. Oligonucleotide-modified gold nanoparticles for intracellular gene regulation. Science 312, 1027–1030 (2006).

  21. 21.

    Ding, Y. et al. Gold nanoparticles for nucleic acid delivery. Mol. Ther. 22, 1075–1083 (2014).

  22. 22.

    Pan, Y. et al. Size-dependent cytotoxicity of gold nanoparticles. Small 3, 1941–1949 (2007).

  23. 23.

    Alkilany, A. M. & Murphy, C. J. Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? J. Nanopart. Res. 12, 2313–2333 (2010).

  24. 24.

    Lewinski, N., Colvin, V. & Drezek, R. Cytotoxicity of nanoparticles. Small 4, 26–49 (2008).

  25. 25.

    Turkevich, J., Stevenson, P. C. & Hillier, J. A study of the nucleation and growth processes in the synthesis of colloidal gold. Discuss. Faraday Soc. 11, 55–75 (1951).

  26. 26.

    Yamano, T. et al. Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell 165, 949–962 (2016).

  27. 27.

    Lee, K. et al. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. eLife 6, e25312 (2017).

  28. 28.

    Lopalco, L. CCR5: From natural resistance to a new anti-HIV strategy. Viruses 2, 574–600 (2010).

  29. 29.

    Akinsheye, I. et al. Fetal hemoglobin in sickle cell anemia. Blood 118, 19–27 (2011).

  30. 30.

    Traxler, E. A. et al. A genome-editing strategy to treat beta-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat. Med. 22, 987–990 (2016).

  31. 31.

    Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

  32. 32.

    Mariam, J., Sivakami, S. & Dongre, P. M. Albumin corona on nanoparticles - a strategic approach in drug delivery. Drug Deliv. 23, 2668–2676 (2016).

  33. 33.

    Yin, H. et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, 1179–1187 (2017).

  34. 34.

    Helgason, C. D. & Miller, C. L. (eds) Basic Cell Culture Protocols 4th edn (Humana, 2013).

  35. 35.

    Notta, F. et al. Distinct routes of lineage development reshape the human blood hierarchy across ontogeny. Science 351, aab2116 (2016).

  36. 36.

    Xiong, X., Chen, M., Lim, W. A., Zhao, D. & Qi, L. S. CRISPR/Cas9 for human genome engineering and disease research. Annu. Rev. Genomics Hum. Genet. 17, 131–154 (2016).

  37. 37.

    Benjaminsen, R. V., Mattebjerg, M. A., Henriksen, J. R., Moghimi, S. M. & Andresen, T. L. The possible “proton sponge” effect of polyethylenimine (PEI) does not include change in lysosomal pH. Mol. Ther. 21, 149–157 (2013).

  38. 38.

    Reza, S., Ilyas, O., Gurkan, O. & Kezban, U. Functionalized gold nanoparticles manifested as potent carriers for nucleolar targeting. Nanotechnology 28, 025103 (2017).

  39. 39.

    Nakade, S., Yamamoto, T. & Sakuma, T. Cas9, Cpf1 and C2c1/2/3―What’s next? Bioengineered 8, 265–273 (2017).

  40. 40.

    de Araújo, R. F.Jr et al. Anti-inflammatory, analgesic and anti-tumor properties of gold nanoparticles. Pharmacol. Rep. 69, 119–129 (2017).

  41. 41.

    Xu, L. et al. CRISPR/Cas9-mediated CCR5 ablation in human hematopoietic stem/progenitor cells confers HIV-1 resistance in vivo. Mol. Ther. 25, 1782–1789 (2017).

  42. 42.

    Radtke, S. et al. A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates. Sci. Transl. Med. 9, eaan1145 (2017).

  43. 43.

    Masiuk, K. E. et al. Improving gene therapy efficiency through the enrichment of human hematopoietic stem cells. Mol. Ther. 25, 2163–2175 (2017).

  44. 44.

    Charpentier, M. et al. CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair. Nat. Commun. 9, 1133 (2018).

  45. 45.

    McMahon, M. A., Prakash, T. P., Cleveland, D. W., Bennett, C. F. & Rahdar, M. Chemically modified Cpf1-CRISPR RNAs mediate efficient genome editing in mammalian cells. Mol. Ther. 26, 1228–1240 (2018).

  46. 46.

    Kimberland, M. L. et al. Strategies for controlling CRISPR/Cas9 off-target effects and biological variations in mammalian genome editing experiments. J. Biotechnol. 284, 91–101 (2018).

  47. 47.

    Glass, Z., Lee, M., Li, Y. & Xu, Q. Engineering the delivery system for CRISPR-based genome editing. Trends Biotechnol. 36, 173–185 (2018).

  48. 48.

    Lino, C. A., Harper, J. C., Carney, J. P. & Timlin, J. A. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv. 25, 1234–1257 (2018).

  49. 49.

    Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).

  50. 50.

    Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017).

  51. 51.

    Shahbazi, R. et al. Modified gold-based siRNA nanotherapeutics for targeted therapy of triple-negative breast cancer. Nanomedicine 12, 1961–1973 (2017).

  52. 52.

    Booth, D. S., Avila-Sakar, A. & Cheng, Y. Visualizing proteins and macromolecular complexes by negative stain EM: from grid preparation to image acquisition. J. Vis. Exp. 58, 3227 (2011).

  53. 53.

    Adair, J. E. et al. Semi-automated closed system manufacturing of lentivirus gene-modified haematopoietic stem cells for gene therapy. Nat. Commun. 7, 13173 (2016).

  54. 54.

    Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

  55. 55.

    Zhang, J., Kobert, K., Flouri, T. & Stamatakis, A. PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 30, 614–620 (2014).

  56. 56.

    Needleman, S. B. & Wunsch, C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48, 443–453 (1970).

  57. 57.

    Kruskal J. B. in Time Warps, String Edits, and Macromolecules: The theory and practice of sequence comparison (eds Sankoff, D. & Kruskal, J. B.) 1–44 (Addison-Wesley, 1983).

  58. 58.

    Haworth, K. G. et al. In vivo murine-matured human CD3+ cells as a preclinical model for T cell-based immunotherapies. Mol. Ther. Methods Clin. Dev. 6, 17–30 (2017).

Download references


We thank the healthy donors who submitted to mobilization and leukapheresis collection. We thank the laboratory of M. Zhang at the University of Washington for access to and use of Nanosizer equipment. We extend a special thanks to H. Crawford for assistance in manuscript preparation, and J. Chen and C. Ironside for excellent support in animal studies. This work was primarily supported by funds to J.E.A. from the Fred Hutch including Development and Evergreen awards, and the Hartwell Foundation. This research was also funded in part through a pilot study award to R.S. from the NIDDK Cooperative Center of Excellence in Hematology grant U54 DK106829. All shared resources used in this study were supported by the NIH/NCI Cancer Center Support Grant P30 CA015704. H.-P.K. is a Markey Molecular Medicine Investigator, the inaugural recipient of the José Carreras/E. Donnall Thomas Endowed Chair for Cancer Research and the Fred Hutch Endowed Chair for Cell and Gene Therapy.

Author information

R.S. and J.E.A. designed the study. R.S., G.S.-H., J.L.R., K.G.H. and O.H. performed the experiments, and generated data and figures. S.K. isolated primary human CD34+ cells from donor products. R.S. and J.E.A. analysed data. R.S., G.S.-H., S.K., K.G.H., O.H. and J.E.A. reviewed and interpreted data. H.-P.K. funded K.G.H. and O.H, served as the IACUC protocol Principal Investigator and provided the Cas9 protein used in the study. R.S. and J.E.A. funded the study and wrote the manuscript. All authors reviewed and edited the final manuscript.

Competing interests

H.-P.K., O.H. and J.E.A. received licensing revenue from Rocket Pharmaceuticals for research unrelated to this manuscript. K.G.H. is employed by and holds equity in Nohla Therapeutics. The other authors declare no competing interests associated with this work. J.E.A. and R.S. are co-inventors on US patent WO2018226762A1 entitled ‘Genomic safe harbors for genetic therapies in human stem cells and engineered nanoparticles to provide targeted genetic therapies.’.

Correspondence to Jennifer E. Adair.

Supplementary information

  1. Supplementary Information

    Supplementary Figs. 1–13 and Tables 1 and 2.

  2. Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
Fig. 1: Layer-by-layer conjugation of CRISPR components onto AuNPs.
Fig. 2: AuNP/CRISPR can deliver CRISPR components to the nucleus of HSPCs.
Fig. 3: Optimization of HDR conditions and optimal editing dosage.
Fig. 4: AuNP/CRISPR nanoformulations carrying Cpf1 outperform Cas9 in terms of HDR.
Fig. 5: AuNP treatment enhanced HSPC engraftment in neonatal immune-deficient mice.
Fig. 6: Persistent editing levels after engraftment.