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.

  • Review Article
  • Published:

Therapeutic genome editing: prospects and challenges

Subjects

Abstract

Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.

This is a preview of subscription content, access via your institution

Access options

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

Figure 1: Types of therapeutic genome modifications.
Figure 2: Factors influencing therapeutic efficacy.
Figure 3: Ex vivo versus in vivo editing therapy.

Similar content being viewed by others

References

  1. Lander, E.S. Initial impact of the sequencing of the human genome. Nature 470, 187–197 (2011).

    CAS  Google Scholar 

  2. Thoene, J.G. Small Molecule Therapy for Genetic Disease (Cambridge University Press, 2010).

    Google Scholar 

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

    CAS  Google Scholar 

  4. Vaishnaw, A.K. et al. A status report on RNAi therapeutics. Silence 1, 14 (2010).

    Google Scholar 

  5. Gaspar, H.B. et al. Long-term persistence of a polyclonal T cell repertoire after gene therapy for X-linked severe combined immunodeficiency. Sci. Transl. Med. 3, 97ra79 (2011).

    Google Scholar 

  6. Howe, S.J. et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J. Clin. Invest. 118, 3143–3150 (2008).

    Article  CAS  Google Scholar 

  7. Aiuti, A. et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science 341, 1233151 (2013).

    Google Scholar 

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

    CAS  Google Scholar 

  9. Tiemann, K. & Rossi, J.J. RNAi-based therapeutics—current status, challenges and prospects. EMBO Mol. Med. 1, 142–151 (2009).

    CAS  Google Scholar 

  10. Jackson, A.L. & Linsley, P.S. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat. Rev. Drug Discov. 9, 57–67 (2010).

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  12. 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).

    Article  CAS  Google Scholar 

  13. Bogdanove, A.J. & Voytas, D.F. TAL effectors: customizable proteins for DNA targeting. Science 333, 1843–1846 (2011).

    CAS  Google Scholar 

  14. Scharenberg, A.M., Duchateau, P. & Smith, J. Genome engineering with TAL-effector nucleases and alternative modular nuclease technologies. Curr. Gene Ther. 13, 291–303 (2013).

    CAS  Google Scholar 

  15. Hsu, P.D., Lander, E.S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    CAS  Google Scholar 

  16. 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  Google Scholar 

  17. Thierry, A. & Dujon, B. Nested chromosomal fragmentation in yeast using the meganuclease I-Sce I: a new method for physical mapping of eukaryotic genomes. Nucleic Acids Res. 20, 5625–5631 (1992).

    CAS  Google Scholar 

  18. Thierry, A. et al. Cleavage of yeast and bacteriophage T7 genomes at a single site using the rare cutter endonuclease I-Sce I. Nucleic Acids Res. 19, 189–190 (1991).

    CAS  Google Scholar 

  19. Smith, J. et al. A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Res. 34, e149 (2006).

    Google Scholar 

  20. Boissel, S. et al. megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Nucleic Acids Res. 42, 2591–2601 (2014).

    CAS  Google Scholar 

  21. Kim, Y.G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156–1160 (1996).

    CAS  Google Scholar 

  22. Wolfe, S.A., Nekludova, L. & Pabo, C.O. DNA recognition by Cys2His2 zinc finger proteins. Annu. Rev. Biophys. Biomol. Struct. 29, 183–212 (2000).

    CAS  Google Scholar 

  23. Bibikova, M., Beumer, K., Trautman, J.K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003).

    CAS  Google Scholar 

  24. Bibikova, M., Golic, M., Golic, K.G. & Carroll, D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–1175 (2002).

    CAS  Google Scholar 

  25. Miller, J., McLachlan, A.D. & Klug, A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 4, 1609–1614 (1985).

    CAS  Google Scholar 

  26. Miller, J.C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).

    CAS  Google Scholar 

  27. Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

    CAS  Google Scholar 

  28. Porteus, M.H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003).

    Google Scholar 

  29. Smith, J. et al. Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res. 28, 3361–3369 (2000).

    CAS  Google Scholar 

  30. Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).

    CAS  Google Scholar 

  31. Moscou, M.J. & Bogdanove, A.J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).

    CAS  Google Scholar 

  32. Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010).

    CAS  Google Scholar 

  33. Miller, J.C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).

    CAS  Google Scholar 

  34. Mahfouz, M.M. et al. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc. Natl. Acad. Sci. USA 108, 2623–2628 (2011).

    CAS  Google Scholar 

  35. Li, T. et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 39, 359–372 (2011).

    Google Scholar 

  36. Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005).

    CAS  Google Scholar 

  37. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).

    CAS  Google Scholar 

  38. Garneau, J.E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010).

    CAS  Google Scholar 

  39. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).

    CAS  Google Scholar 

  40. Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275–9282 (2011).

    CAS  Google Scholar 

  41. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Google Scholar 

  42. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109, E2579–E2586 (2012).

    CAS  Google Scholar 

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

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  45. Isalan, M. Zinc-finger nucleases: how to play two good hands. Nat. Methods 9, 32–34 (2012).

    CAS  Google Scholar 

  46. Sun, N. & Zhao, H. Transcription activator-like effector nucleases (TALENs): a highly efficient and versatile tool for genome editing. Biotechnol. Bioeng. 110, 1811–1821 (2013).

    CAS  Google Scholar 

  47. 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).

    CAS  Google Scholar 

  48. Genovese, P. et al. Targeted genome editing in human repopulating haematopoietic stem cells. Nature 510, 235–240 (2014).

    CAS  Google Scholar 

  49. Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221 (2011).

    CAS  Google Scholar 

  50. Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).

    CAS  Google Scholar 

  51. Hentze, M.W. & Kulozik, A.E. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 96, 307–310 (1999).

    CAS  Google Scholar 

  52. Choulika, A., Perrin, A., Dujon, B. & Nicolas, J.F. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol. Cell. Biol. 15, 1968–1973 (1995).

    CAS  Google Scholar 

  53. Bibikova, M. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell. Biol. 21, 289–297 (2001).

    CAS  Google Scholar 

  54. Krejci, L., Altmannova, V., Spirek, M. & Zhao, X. Homologous recombination and its regulation. Nucleic Acids Res. 40, 5795–5818 (2012).

    CAS  Google Scholar 

  55. Plessis, A., Perrin, A., Haber, J.E. & Dujon, B. Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics 130, 451–460 (1992).

    CAS  Google Scholar 

  56. Rudin, N., Sugarman, E. & Haber, J.E. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics 122, 519–534 (1989).

    CAS  Google Scholar 

  57. Liu, R. et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86, 367–377 (1996).

    CAS  Google Scholar 

  58. Cohen, J. et al. Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9. Nat. Genet. 37, 161–165 (2005).

    CAS  Google Scholar 

  59. Jonsson, T. et al. A mutation in APP protects against Alzheimer's disease and age-related cognitive decline. Nature 488, 96–99 (2012).

    CAS  Google Scholar 

  60. Lombardo, A. et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat. Methods 8, 861–869 (2011).

    CAS  Google Scholar 

  61. 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  Google Scholar 

  62. Barzel, A. et al. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature doi:10.1038/nature13864 (2014).

  63. Maude, S.L. et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N. Engl. J. Med. 371, 1507–1517 (2014).

    Google Scholar 

  64. Hu, W. et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc. Natl. Acad. Sci. USA 111, 11461–11466 (2014).

    CAS  Google Scholar 

  65. Lin, S.R. et al. The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo. Mol. Ther. Nucleic Acids 3, e186 (2014).

    CAS  Google Scholar 

  66. Bloom, K., Ely, A., Mussolino, C., Cathomen, T. & Arbuthnot, P. Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases. Mol. Ther. 21, 1889–1897 (2013).

    CAS  Google Scholar 

  67. Cradick, T.J., Keck, K., Bradshaw, S., Jamieson, A.C. & McCaffrey, A.P. Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs. Mol. Ther. 18, 947–954 (2010).

    CAS  Google Scholar 

  68. Weber, N.D. et al. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication. PLoS ONE 9, e97579 (2014).

    Google Scholar 

  69. Holt, N. et al. Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat. Biotechnol. 28, 839–847 (2010).

    CAS  Google Scholar 

  70. Perez, E.E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008).

    CAS  Google Scholar 

  71. Hacein-Bey-Abina, S. et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N. Engl. J. Med. 346, 1185–1193 (2002).

    CAS  Google Scholar 

  72. Gaspar, H.B. et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 364, 2181–2187 (2004).

    CAS  Google Scholar 

  73. Bousso, P. et al. Diversity, functionality, and stability of the T cell repertoire derived in vivo from a single human T cell precursor. Proc. Natl. Acad. Sci. USA 97, 274–278 (2000).

    CAS  Google Scholar 

  74. Malech, H.L. et al. Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease. Proc. Natl. Acad. Sci. USA 94, 12133–12138 (1997).

    CAS  Google Scholar 

  75. Kang, H.J. et al. Retroviral gene therapy for X-linked chronic granulomatous disease: results from phase I/II trial. Mol. Ther. 19, 2092–2101 (2011).

    CAS  Google Scholar 

  76. Löfqvist, T., Nilsson, I.M., Berntorp, E. & Pettersson, H. Haemophilia prophylaxis in young patients—a long-term follow-up. J. Intern. Med. 241, 395–400 (1997).

    Google Scholar 

  77. Kaushansky, K. & Williams, W.J. Williams Hematology (McGraw-Hill Medical, New York, 2010).

    Google Scholar 

  78. Rothkamm, K., Kruger, I., Thompson, L.H. & Lobrich, M. Pathways of DNA double-strand break repair during the mammalian cell cycle. Mol. Cell. Biol. 23, 5706–5715 (2003).

    CAS  Google Scholar 

  79. Sharma, S. Age-related nonhomologous end joining activity in rat neurons. Brain Res. Bull. 73, 48–54 (2007).

    Google Scholar 

  80. Ciccia, A. & Elledge, S.J. The DNA damage response: making it safe to play with knives. Mol. Cell 40, 179–204 (2010).

    CAS  Google Scholar 

  81. 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  Google Scholar 

  82. Beumer, K.J., Trautman, J.K., Mukherjee, K. & Carroll, D. Donor DNA utilization during gene targeting with zinc-finger nucleases. G3 http://dx.doi.org/10.1534/g3.112.005439 (2013).

  83. Moynahan, M.E. & Jasin, M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat. Rev. Mol. Cell Biol. 11, 196–207 (2010).

    CAS  Google Scholar 

  84. Orlando, S.J. et al. Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res. 38, e152 (2010).

    Google Scholar 

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

    CAS  Google Scholar 

  86. Miller, D.G. et al. Gene targeting in vivo by adeno-associated virus vectors. Nat. Biotechnol. 24, 1022–1026 (2006).

    CAS  Google Scholar 

  87. Beumer, K.J. et al. Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105, 19821–19826 (2008).

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  89. Bunn, H.F. & Aster, J. Pathophysiology of Blood Disorders (McGraw-Hill, New York, 2011).

    Google Scholar 

  90. Kotterman, M.A. & Schaffer, D.V. Engineering adeno-associated viruses for clinical gene therapy. Nat. Rev. Genet. 15, 445–451 (2014).

    CAS  Google Scholar 

  91. Nguyen, T.H. & Ferry, N. Liver gene therapy: advances and hurdles. Gene Ther. 11 (suppl. 1), S76–S84 (2004).

    CAS  Google Scholar 

  92. Boye, S.E., Boye, S.L., Lewin, A.S. & Hauswirth, W.W. A comprehensive review of retinal gene therapy. Mol. Ther. 21, 509–519 (2013).

    CAS  Google Scholar 

  93. Bessis, N., GarciaCozar, F.J. & Boissier, M.C. Immune responses to gene therapy vectors: influence on vector function and effector mechanisms. Gene Ther. 11 (suppl. 1), S10–S17 (2004).

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  95. Mingozzi, F. & High, K.A. Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood 122, 23–36 (2013).

    CAS  Google Scholar 

  96. Lamers, C.H. et al. Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells. Blood 117, 72–82 (2011).

    CAS  Google Scholar 

  97. Hütter, G. et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N. Engl. J. Med. 360, 692–698 (2009).

    Google Scholar 

  98. 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  Google Scholar 

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

    CAS  Google Scholar 

  100. Weller, S.K. & Sawitzke, J.A. Recombination promoted by DNA viruses: phage lambda to herpes simplex virus. Annu. Rev. Microbiol. 68, 237–258 (2014).

    CAS  Google Scholar 

  101. Maresca, M., Lin, V.G., Guo, N. & Yang, Y. Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining. Genome Res. 23, 539–546 (2013).

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  103. Nakade, S. et al. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat. Commun. 5, 5560 (2014).

    CAS  Google Scholar 

  104. Pattanayak, V., Ramirez, C.L., Joung, J.K. & Liu, D.R. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat. Methods 8, 765–770 (2011).

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  106. Guilinger, J.P. et al. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat. Methods 11, 429–435 (2014).

    CAS  Google Scholar 

  107. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

    CAS  Google Scholar 

  108. Cho, S.W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141 (2014).

    CAS  Google Scholar 

  109. Mali, P. et al. Barcoding cells using cell-surface programmable DNA-binding domains. Nat. Methods 10, 403–406 (2013).

    CAS  Google Scholar 

  110. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).

    CAS  Google Scholar 

  111. Veres, A. et al. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell 15, 27–30 (2014).

    CAS  Google Scholar 

  112. Tan, E.P., Li, Y., Del Castillo Velasco-Herrera, M., Yusa, K. & Bradley, A. Off-target assessment of CRISPR-Cas9 guiding RNAs in human iPS and mouse ES cells. Genesis doi:10.1002/dvg.22835 (2014).

  113. Yang, L. et al. Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells. Nat. Commun. 5, 5507 (2014).

    CAS  Google Scholar 

  114. Crosetto, N. et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods 10, 361–365 (2013).

    CAS  Google Scholar 

  115. Tsai, S.Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. doi:10.1038/nbt.3117 (2014).

  116. Frock, R.L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. doi:10.1038/nbt.3101 (2014).

  117. 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 (2014).

    CAS  Google Scholar 

  118. 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  Google Scholar 

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

    CAS  Google Scholar 

  120. Lisowski, L. et al. Ribosomal DNA integrating rAAV-rDNA vectors allow for stable transgene expression. Mol. Ther. 20, 1912–1923 (2012).

    CAS  Google Scholar 

  121. Shi, Y., Falahati, R., Zhang, J., Flebbe-Rehwaldt, L. & Gaensler, K.M. Role of antigen-specific regulatory CD4+CD25+ T cells in tolerance induction after neonatal IP administration of AAV-hF.IX. Gene Ther. 20, 987–996 (2013).

    CAS  Google Scholar 

  122. Paulk, N.K., Loza, L.M., Finegold, M.J. & Grompe, M. AAV-mediated gene targeting is significantly enhanced by transient inhibition of nonhomologous end joining or the proteasome in vivo. Hum. Gene Ther. 23, 658–665 (2012).

    CAS  Google Scholar 

  123. Zuris, J.A. et al. Cationic lipid–mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. doi:10.1038/nbt.3081 (2014).

  124. 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  Google Scholar 

  125. Wirth, T., Parker, N. & Yla-Herttuala, S. History of gene therapy. Gene 525, 162–169 (2013).

    CAS  Google Scholar 

  126. Samulski, R.J. & Muzyczka, N. AAV-mediated gene therapy for research and therapeutic purposes. Annu. Rev. Virol. 1, 427–451 (2014).

    Google Scholar 

  127. Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. doi:10.1038/nbt.3055 (2014).

  128. Moore, R. et al. CRISPR-based self-cleaving mechanism for controllable gene delivery in human cells. Nucleic Acids Res. doi:10.1093/nar/gku1326 (2014).

    CAS  Google Scholar 

  129. Kormann, M.S. et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 29, 154–157 (2011).

    CAS  Google Scholar 

  130. Riechmann, L., Clark, M., Waldmann, H. & Winter, G. Reshaping human antibodies for therapy. Nature 332, 323–327 (1988).

    CAS  Google Scholar 

  131. Ye, L. et al. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Delta32 mutation confers resistance to HIV infection. Proc. Natl. Acad. Sci. USA 111, 9591–9596 (2014).

    CAS  Google Scholar 

  132. Ousterout, D.G. et al. Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients. Mol. Ther. 21, 1718–1726 (2013).

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  134. Wu, Y. et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13, 659–662 (2013).

    CAS  Google Scholar 

  135. 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  Google Scholar 

  136. Crosby, T. et al. Loss-of-function mutations in APOC3, triglycerides, and coronary disease. N. Engl. J. Med. 371, 22–31 (2014).

    Google Scholar 

  137. Bauer, D.E. et al. An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science 342, 253–257 (2013).

    CAS  Google Scholar 

  138. Menzel, S. et al. A QTL influencing F cell production maps to a gene encoding a zinc-finger protein on chromosome 2p15. Nat. Genet. 39, 1197–1199 (2007).

    CAS  Google Scholar 

  139. Xu, J. et al. Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing. Science 334, 993–996 (2011).

    CAS  Google Scholar 

  140. Pennisi, E. The Biology of Genomes. Disease risk links to gene regulation. Science 332, 1031 (2011).

    Google Scholar 

  141. Kennedy, E.M. et al. Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells using a bacterial CRISPR/Cas RNA-guided endonuclease. J. Virol. doi:10.1128/JVI.01879-14 (2014).

Download references

Acknowledgements

The authors would like to thank J. Gootenberg, O. Abudayyeh, F. Ran and C. Men for critical reading of the manuscript, and all members of the Zhang lab for helpful discussions. D.B.T.C. is supported by award number T32GM007753 from the National Institute of General Medical Sciences. R.J.P. is supported by a National Science Foundation (NSF) Graduate Research Fellowship under grant number 1122374. F.Z. is supported by the National Institute of Mental Health through a US National Institutes of Health (NIH) Director's Pioneer Award (DP1-MH100706); the National Institute of Neurological Disorders and Stroke through an NIH Transformative R01 grant (R01-NS 07312401); an NSF Waterman Award; and the Keck, Damon Runyon, Searle Scholars, Klingenstein, Vallee, Merkin, and Simons Foundations. F.Z. is also supported by Bob Metcalfe. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the NIH.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Feng Zhang.

Ethics declarations

Competing interests

F.Z. is a founder and scientific advisor of Editas Medicine and a scientific advisor of Horizon Discovery.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cox, D., Platt, R. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat Med 21, 121–131 (2015). https://doi.org/10.1038/nm.3793

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.3793

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research