Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Delivery technologies for genome editing

Key Points

  • Genome-editing systems have remarkable potential to treat genetic diseases. However, one of the major challenges facing their implementation is the safe and efficient intracellular delivery of genome-editing biomacromolecules, including nucleases and nucleic acids.

  • Nanoparticles encapsulating genome-editing biomacromolecules must be endocytosed by the cell of interest and reach the nucleus or cytoplasm to function; additional extracellular barriers are present for in vivo delivery.

  • Physical methods for in vitro and ex vivo delivery of genome-editing tools include electroporation, membrane deformation and microinjection.

  • Viral vectors are widely used to deliver DNA for genome editing and are typically integrase-defective lentiviral vectors (IDLVs), adenoviruses and adeno-associated viruses (AAVs); AAVs seem to be the most popular vector for in vivo applications.

  • Non-viral nanoparticles, most often made from synthetic and cationic lipid or polymer delivery materials, can be used to deliver genome-editing tools in vitro, ex vivo and in vivo, sometimes in combination with viral vectors.

  • At the time of writing, the genome-editing industry is in its infancy and includes ongoing and completed phase I and phase II clinical trials.

  • In addition to selecting an effective delivery material, scientists must consider safety (that is, off-target effects, immunogenicity and mutagenesis), the required amount of genomic modification for therapeutic benefit, and the duration of effects.

Abstract

With the recent development of CRISPR technology, it is becoming increasingly easy to engineer the genome. Genome-editing systems based on CRISPR, as well as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs), are becoming valuable tools for biomedical research, drug discovery and development, and even gene therapy. However, for each of these systems to effectively enter cells of interest and perform their function, efficient and safe delivery technologies are needed. This Review discusses the principles of biomacromolecule delivery and gene editing, examines recent advances and challenges in non-viral and viral delivery methods, and highlights the status of related clinical trials.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: The mechanisms of genome editing and DSB repair.
Figure 2: Barriers to delivery of genome-editing components.
Figure 3: Physical methods used for delivery of genome-editing biomacromolecules.
Figure 4: Non-viral nanoparticles for delivery of genome-editing biomacromolecules.

References

  1. Boycott, K. M., Vanstone, M. R., Bulman, D. E. & MacKenzie, A. E. Rare-disease genetics in the era of next-generation sequencing: discovery to translation. Nat. Rev. Genet. 14, 681–691 (2013).

    CAS  PubMed  Google Scholar 

  2. Pharmaceutical Research and Manufacturers of America. Medicines In Development. Rare Diseases: A Report On Orphan Drugs In The Pipeline Presented By America's Biopharmaceutical Research Companies. PhRMA.org http://phrma-docs.phrma.org/sites/default/files/pdf/Rare_Diseases_2013.pdf (2013).

  3. Elborn, J. S. Cystic fibrosis. Lancet 388, 2519–2531 (2016).

    CAS  PubMed  Google Scholar 

  4. Yang, Q. Small molecule therapy for genetic diseases. Yale J. Biol. Med. 85, 161–162 (2012).

    PubMed Central  Google Scholar 

  5. O'Connor, T. P. & Crystal, R. G. Genetic medicines: treatment strategies for hereditary disorders. Nat. Rev. Genet. 7, 261–276 (2006).

    CAS  PubMed  Google Scholar 

  6. Winkel, L. P. et al. Enzyme replacement therapy in late-onset Pompe's disease: a three-year follow-up. Ann. Neurol. 55, 495–502 (2004).

    CAS  PubMed  Google Scholar 

  7. Srivastava, A. Dose and response in haemophilia — optimization of factor replacement therapy. Br. J. Haematol. 127, 12–25 (2004).

    PubMed  Google Scholar 

  8. Holz, F. G., Schmitz-Valckenberg, S. & Fleckenstein, M. Recent developments in the treatment of age-related macular degeneration. J. Clin. Invest. 124, 1430–1438 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Gorzelany, J. A. & de Souza, M. P. Protein replacement therapies for rare diseases: a breeze for regulatory approval? Sci. Transl Med. 5, 178fs10 (2013).

    PubMed  Google Scholar 

  10. Yin, H. et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555 (2014).

    CAS  PubMed  Google Scholar 

  11. Kay, M. A. State-of-the-art gene-based therapies: the road ahead. Nat. Rev. Genet. 12, 316–328 (2011).

    CAS  PubMed  Google Scholar 

  12. Mansoor, M. & Melendez, A. J. Advances in antisense oligonucleotide development for target identification, validation, and as novel therapeutics. Gene Regul. Syst. Bio. 2, 275–295 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Castanotto, D. & Rossi, J. J. The promises and pitfalls of RNA-interference-based therapeutics. Nature 457, 426–433 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Baum, C., Kustikova, O., Modlich, U., Li, Z. & Fehse, B. Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors. Hum. Gene Ther. 17, 253–263 (2006).

    CAS  PubMed  Google Scholar 

  15. Herzog, R. W. Hemophilia gene therapy: caught between a cure and an immune response. Mol. Ther. 23, 1411–1412 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Herzog, R. W., Davidoff, A. M., Markusic, D. M. & Nathwani, A. C. AAV vector biology in primates: finding the missing link? Mol. Ther. 19, 1923–1924 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Wang, L. et al. AAV8-mediated hepatic gene transfer in infant rhesus monkeys (Macaca mulatta). Mol. Ther. 19, 2012–2020 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Naldini, L. Gene therapy returns to centre stage. Nature 526, 351–360 (2015).

    CAS  PubMed  Google Scholar 

  19. Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

    PubMed  Google Scholar 

  20. Stoddard, B. L. Homing endonucleases: from microbial genetic invaders to reagents for targeted DNA modification. Structure 19, 7–15 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

    CAS  PubMed  Google Scholar 

  22. Joung, J. K. & Sander, J. D. TALENs: a widely applicable technology for targeted genome editing. Nat. Rev. Mol. Cell Biol. 14, 49–55 (2013).

    CAS  PubMed  Google Scholar 

  23. Rouet, P., Smih, F. & Jasin, M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell. Biol. 14, 8096–8106 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Lieber, M. R., Ma, Y., Pannicke, U. & Schwarz, K. Mechanism and regulation of human non-homologous DNA end-joining. Nat. Rev. Mol. Cell Biol. 4, 712–720 (2003).

    CAS  PubMed  Google Scholar 

  25. Isken, O. & Maquat, L. E. Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function. Genes Dev. 21, 1833–1856 (2007).

    CAS  PubMed  Google Scholar 

  26. Long, C. et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351, 400–403 (2016).

    CAS  PubMed  Google Scholar 

  27. Long, C. et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345, 1184–1188 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Nelson, C. E. et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351, 403–407 (2016).

    CAS  PubMed  Google Scholar 

  29. Tabebordbar, M. et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351, 407–411 (2016).

    CAS  PubMed  Google Scholar 

  30. Xu, L. et al. CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice. Mol. Ther. 24, 564–569 (2016). References 26, 28, 29 and 30 demonstrate the use of viral vectors to correct disease-causing mutations in a mouse model of Duchenne muscular dystrophy.

    PubMed  PubMed Central  Google Scholar 

  31. Sadelain, M., Papapetrou, E. P. & Bushman, F. D. Safe harbours for the integration of new DNA in the human genome. Nat. Rev. Cancer 12, 51–58 (2012).

    CAS  Google Scholar 

  32. Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221 (2011). This study is the first to demonstrate that ZFN-mediated gene correction can be achieved in vivo and reverse the disease phenotype.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014). This study is the first time to demonstrate that CRISPR can correct a disease mutation in vivo and reverse disease symptoms.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Oakes, B. L. et al. Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing. Nat. Commun. 7, 10194 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Bhakta, M. S. et al. Highly active zinc-finger nucleases by extended modular assembly. Genome Res. 23, 530–538 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Wilson, K. A. et al. Expanding the repertoire of target sites for zinc finger nuclease-mediated genome modification. Mol. Ther. Nucleic Acids 2, e88 (2013).

    PubMed  PubMed Central  Google Scholar 

  37. Shukla, V. K. et al. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459, 437–441 (2009).

    CAS  PubMed  Google Scholar 

  38. Kim, H. & Kim, J.-S. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321–334 (2014).

    CAS  PubMed  Google Scholar 

  39. Kim, Y. et al. A library of TAL effector nucleases spanning the human genome. Nat. Biotechnol. 31, 251–258 (2013).

    CAS  PubMed  Google Scholar 

  40. Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2015).

    Google Scholar 

  41. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011).

    CAS  PubMed  Google Scholar 

  43. Sander, J. D. et al. In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites. Nucleic Acids Res. 41, e181 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    CAS  PubMed  Google Scholar 

  45. Frock, R. L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179–186 (2015).

    CAS  PubMed  Google Scholar 

  46. Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).

    CAS  PubMed  Google Scholar 

  47. Wang, X. et al. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat. Biotechnol. 33, 175–178 (2015).

    CAS  PubMed  Google Scholar 

  48. Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Tsai, S. Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Guilinger, J. P., Thompson, D. B. & Liu, D. R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2013).

    Google Scholar 

  53. Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

    CAS  PubMed  Google Scholar 

  54. Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detec table genome-wide off-target effects. Nature 529, 490–495 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Bolukbasi, M. F. et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat. Methods 12, 1150–1156 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Sollu, C. et al. Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion. Nucleic Acids Res. 38, 8269–8276 (2010).

    PubMed  PubMed Central  Google Scholar 

  57. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Senis, E. et al. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol. J. 9, 1402–1412 (2014).

    CAS  PubMed  Google Scholar 

  60. Genovese, P. et al. Targeted genome editing in human repopulating haematopoietic stem cells. Nature 510, 235–240 (2014). This study demonstrates that gene-edited HSCs can sustain normal haematopoiesis.

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Wang, D. et al. Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum. Gene Ther. 26, 432–442 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl Acad. Sci. USA 112, 10437–10442 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Mandal, P. K. et al. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15, 643–652 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Han, X. et al. CRISPR–Cas9 delivery to hard-to-transfect cells via membrane deformation. Sci. Adv. 1, e1500454 (2015).

    PubMed  PubMed Central  Google Scholar 

  65. Sharei, A. et al. A vector-free microfluidic platform for intracellular delivery. Proc. Natl Acad. Sci. USA 110, 2082–2087 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2014). This study reports that proteins fused to negatively charged domains can be intracellularly delivered by cationic lipids.

    PubMed  PubMed Central  Google Scholar 

  67. Liang, X. et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J. Biotechnol. 208, 44–53 (2015).

    CAS  PubMed  Google Scholar 

  68. Ramakrishna, S. et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24, 1020–1027 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Sun, W. et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew. Chem. Int. Ed. 54, 12029–12033 (2015).

    CAS  Google Scholar 

  70. Morris, E. C. & Stauss, H. J. Optimizing T cell receptor gene therapy for hematologic malignancies. Blood 127, 3305–3311 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Braun, C. J. et al. Gene therapy for Wiskott–Aldrich syndrome — long-term efficacy and genotoxicity. Sci. Transl Med. 6, 227ra33 (2014).

    PubMed  Google Scholar 

  72. Bankiewicz, K. S. et al. Long-term clinical improvement in MPTP-lesioned primates after gene therapy with AAV-hAADC. Mol. Ther. 14, 564–570 (2006).

    CAS  PubMed  Google Scholar 

  73. Yin, H. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 34, 328–333 (2016). This study reports that a combination of lipid nanoparticles encapsulating Cas9 mRNA with an AAV encoding a repair donor and an sgRNA induces efficient repair of a disease gene in vivo.

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Yin, H. et al. RNAi-nanoparticulate manipulation of gene expression as a new functional genomics tool in the liver. J. Hepatol. 64, 899–907 (2016).

    CAS  PubMed  Google Scholar 

  75. Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33, 102–106 (2014).

    PubMed  PubMed Central  Google Scholar 

  76. Lombardo, A. et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat. Biotechnol. 25, 1298–1306 (2007).

    CAS  PubMed  Google Scholar 

  77. De Ravin, S. S. et al. Targeted gene addition in human CD34 hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat. Biotechnol. 34, 424–429 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Wang, J. et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat. Biotechnol. 33, 1256–1263 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Provasi, E. et al. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat. Med. 18, 807–815 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Sather, B. D. et al. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Sci. Transl Med. 7, 307ra156 (2015).

    PubMed  PubMed Central  Google Scholar 

  81. Holkers, M. et al. Adenoviral vector DNA for accurate genome editing with engineered nucleases. Nat. Methods 11, 1051–1057 (2014).

    CAS  PubMed  Google Scholar 

  82. Weissman, I. L. & Shizuru, J. A. The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases. Blood 112, 3543–3553 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Khalil, D. N., Smith, E. L., Brentjens, R. J. & Wolchok, J. D. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat. Rev. Clin. Oncol. 13, 273–290 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. DeKelver, R. C. et al. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res. 20, 1133–1142 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Cohen, C. J. et al. Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond. Cancer Res. 67, 3898–3903 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Beane, J. D. et al. Clinical scale zinc finger nuclease-mediated gene editing of PD-1 in tumor infiltrating lymphocytes for the treatment of metastatic melanoma. Mol. Ther. 23, 1380–1390 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Torikai, H. et al. Toward eliminating HLA class I expression to generate universal cells from allogeneic donors. Blood 122, 1341–1349 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Frederickson, R. M. A new era of innovation for CAR T-cell therapy. Mol. Ther. 23, 1795–1796 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Wang, J. et al. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery. Nucleic Acids Res. 44, e30 (2016).

    PubMed  Google Scholar 

  90. Wang, X. et al. CRISPR-Cas9 targeting of PCSK9 in human hepatocytes in vivo. Arterioscler. Thromb. Vasc. Biol. 36, 783–786 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Ding, Q. et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ. Res. 115, 488–492 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Anguela, X. M. et al. Robust ZFN-mediated genome editing in adult hemophilic mice. Blood 122, 3283–3287 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Sharma, R. et al. In vivo genome editing of the albumin locus as a platform for protein replacement therapy. Blood 126, 1777–1784 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).

    CAS  PubMed  Google Scholar 

  95. Wright, A. V. et al. Rational design of a split-Cas9 enzyme complex. Proc. Natl Acad. Sci. USA 112, 2984–2989 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  96. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015). This study characterizes smaller Cas9 orthologues, and packages one orthologue and a guide RNA into a single AAV vector to perform in vivo editing.

    CAS  PubMed  PubMed Central  Google Scholar 

  97. Yang, Y. et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat. Biotechnol. 34, 334–338 (2016). This study uses dual AAV vectors to deliver Cas9, a guide RNA and a template DNA to efficiently correct a mutation in the liver.

    CAS  PubMed  PubMed Central  Google Scholar 

  98. Rols, M. P. Mechanism by which electroporation mediates DNA migration and entry into cells and targeted tissues. Methods Mol. Biol. 423, 19–33 (2008).

    CAS  PubMed  Google Scholar 

  99. Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  100. Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015). This study reports that chemical modification of sgRNA enhances genome-editing efficiency in primary cells and stem cells.

    CAS  PubMed  PubMed Central  Google Scholar 

  101. Derdelinckx, J., Berneman, Z. N. & Cools, N. GMP-grade mRNA electroporation of dendritic cells for clinical use. Methods Mol. Biol. 1428, 139–150 (2016).

    CAS  PubMed  Google Scholar 

  102. DiTommaso, T., Gilbert, J., Bernstein, H. & Sharei, A. Vector free genome editing of immune cells for cell therapy. Mol. Ther. 24 (Suppl. 1), S229 (2016).

    Google Scholar 

  103. D'Astolfo, D. S. et al. Efficient intracellular delivery of native proteins. Cell 161, 674–690 (2015).

    CAS  PubMed  Google Scholar 

  104. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. Schwank, G. et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013).

    CAS  PubMed  Google Scholar 

  106. Liu, J., Gaj, T., Patterson, J. T., Sirk, S. J. & Barbas, C. F. III. Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering. PLoS ONE 9, e85755 (2014).

    PubMed  PubMed Central  Google Scholar 

  107. Gaj, T., Guo, J., Kato, Y., Sirk, S. J. & Barbas, C. F. III. Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat. Methods 9, 805–807 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  108. Liu, F., Song, Y. & Liu, D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6, 1258–1266 (1999).

    CAS  PubMed  Google Scholar 

  109. Khorsandi, S. E. et al. Minimally invasive and selective hydrodynamic gene therapy of liver segments in the pig and human. Cancer Gene Ther. 15, 225–230 (2008).

    CAS  PubMed  Google Scholar 

  110. Mahiny, A. J. et al. In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency. Nat. Biotechnol. 33, 584–586 (2015).

    CAS  PubMed  Google Scholar 

  111. Wang, M. et al. Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc. Natl Acad. Sci. USA 113, 2868–2873 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Yang, L. et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science 350, 1101–1104 (2015).

    CAS  PubMed  Google Scholar 

  114. Liang, P. et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6, 363–372 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  115. Baltimore, B. D. et al. A prudent path forward for genomic engineering and germline gene modification. Science 348, 36–38 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT01543152 (2016).

  117. US National Library of Medicine. ClinicalTrials.gov http://www.clinicaltrials.gov/ct2/show/NCT00842634 (2016).

  118. Tebas, P. et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N. Engl. J. Med. 370, 901–910 (2014). This study tests the safety and feasibility of ZFNs targeting CCR5 in patients.

    CAS  PubMed  PubMed Central  Google Scholar 

  119. US National Library of Medicine. ClinicalTrials.gov http://www.clinicaltrials.gov/ct2/show/NCT02225665 (2015).

  120. US National Library of Medicine. ClinicalTrials.gov http://www.clinicaltrials.gov/ct2/show/NCT02500849 (2016).

  121. US National Library of Medicine. ClinicalTrials.gov http://www.clinicaltrials.gov/ct2/show/NCT02695160 (2016).

  122. US National Library of Medicine. ClinicalTrials.gov http://www.clinicaltrials.gov/ct2/show/NCT02702115 (2016).

  123. Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–385 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Mueller, C. et al. Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles. Mol. Ther. 20, 590–600 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. Chapman, J. R., Taylor, M. R. & Boulton, S. J. Playing the end game: DNA double-strand break repair pathway choice. Mol. Cell 47, 497–510 (2012).

    CAS  PubMed  Google Scholar 

  126. Wu, W. H. et al. CRISPR repair reveals causative mutation in a preclinical model of retinitis pigmentosa. Mol. Ther. 24, 1388–1394 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. Miller, D. G., Petek, L. M. & Russell, D. W. Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks. Mol. Cell. Biol. 23, 3550–3557 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  128. Koudelka, K. J., Pitek, A. S., Manchester, M. & Steinmetz, N. F. Virus-based nanoparticles as versatile nanomachines. Annu. Rev. Virol. 2, 379–401 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  129. Heyer, W. D., Ehmsen, K. T. & Liu, J. Regulation of homologous recombination in eukaryotes. Annu. Rev. Genet. 44, 113–139 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  130. Moehle, E. A. et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc. Natl Acad. Sci. USA 104, 3055–3060 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  131. Chen, F. et al. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat. Methods 8, 753–755 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  132. Maruyama, T. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33, 538–542 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. Chu, V. T. et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015).

    CAS  PubMed  Google Scholar 

  134. High, K. H., Nathwani, A., Spencer, T. & Lillicrap, D. Current status of haemophilia gene therapy. Haemophilia 20 (Suppl. 4), 43–49 (2014).

    CAS  PubMed  Google Scholar 

  135. Doerfler, P. A. et al. Targeted approaches to induce immune tolerance for Pompe disease therapy. Mol. Ther. Methods Clin. Dev. 3, 15053 (2016).

    PubMed  PubMed Central  Google Scholar 

  136. Gaj, T., Epstein, B. E. & Schaffer, D. V. Genome engineering using adeno-associated virus: basic and clinical research applications. Mol. Ther. 24, 458–464 (2016).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  138. Cavazzana-Calvo, M. et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288, 669–672 (2000).

    CAS  PubMed  Google Scholar 

  139. Chen, X., Gonçalves, M. A. et al. Engineered viruses as genome editing devices. Mol. Ther. 3, 447–457 (2016).

    Google Scholar 

  140. Waehler, R., Russell, S. J. & Curiel, D. T. Engineering targeted viral vectors for gene therapy. Nat. Rev. Genet. 8, 573–587 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J. E. Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 302, 1073–1080 (2008).

    Google Scholar 

  142. Bouard, D., Alazard-Dany, D., Cosset, F. L., et al. Viral vectors: from virology to transgene expression. Br. J. Pharmacol. 157, 153–165 (2000).

    Google Scholar 

  143. US National Library of Medicine. ClinicalTrials.gov http://www.clinicaltrials.gov/ct2/show/NCT03041324 (2017).

  144. US National Library of Medicine. ClinicalTrials.gov http://www.clinicaltrials.gov/ct2/show/NCT01252641 (2015).

  145. US National Library of Medicine. ClinicalTrials.gov http://www.clinicaltrials.gov/ct2/show/NCT01079325 (2016).

  146. US National Library of Medicine. ClinicalTrials.gov http://www.clinicaltrials.gov/ct2/show/NCT00748501 (2012).

Download references

Acknowledgements

H.Y., K.J.K. and D.G.A. acknowledge funding from the Koch Institute Marble Center for Cancer Nanomedicine and the Cancer Center Support (core) Grant P30-CA14051. H.Y. is supported by Skoltech Center. The authors apologize to those authors whose work was not cited directly owing to space limitations.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Daniel G. Anderson.

Ethics declarations

Competing interests

D.G.A. and H.Y. have applied for patents relating to delivery technologies for genome editing. D.G.A. is a scientific co-founder of CRISPR Therapeutics.

Related links

PowerPoint slides

Glossary

RNA interference

(RNAi). Process by which one strand of double-stranded RNA binds to complementary mRNA and degrades or regulates the mRNA via an enzymatic process, usually resulting in a decrease in the expression of a desired protein.

Antisense oligonucleotides

(ASOs). Short single-stranded DNA or RNA sequences that bind to complementary mRNAs, inhibit translation and/or degrade the targeted mRNA, resulting in a decrease in the expression of a desired protein.

Episomal

DNA that functions without integrating into the genome: for example, a delivered DNA plasmid.

Protospacer adjacent motif

A short, typically 2–6 nucleotide-long region of DNA recognized by the Cas9 protein, located immediately next to the target region for the Cas9 nuclease.

Tropism

The ability of a virus to specifically target particular cells or tissues.

Allogeneic

From the same species but not genetically compatible; that is, will induce an immune response.

Ribonucleoprotein

(RNP). Any biomacromolecule consisting of an RNA in complex with a protein.

Hydrodynamic injection

A rapid, high-volume intravenous infusion.

Monogenic

Under the control of a single gene.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yin, H., Kauffman, K. & Anderson, D. Delivery technologies for genome editing. Nat Rev Drug Discov 16, 387–399 (2017). https://doi.org/10.1038/nrd.2016.280

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrd.2016.280

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing