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.

A guide to genome engineering with programmable nucleases

Key Points

  • Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR (clustered regularly interspaced short palindromic repeat)-associated protein 9 (Cas9) RNA-guided engineered nucleases (RGENs) — enable genome editing in cultured cells, as well as in whole animals and plants. These nucleases are useful for a broad range of applications in biomedical research, medicine and biotechnology.

  • ZFNs and TALENs are composed of DNA-binding proteins and the FokI nuclease domain. RGENs are derived from the type II CRISPR–Cas adaptive immune system in bacteria and are composed of guide RNAs and the Cas9 protein.

  • DNA double-strand breaks (DSBs) introduced by programmable nucleases can be repaired by homology-directed repair, which leads to gene insertion, correction and point mutagenesis, or by erroneous non-homologous end-joining, which results in gene disruptions. The repair of two concurrent DSBs can give rise to chromosomal rearrangements such as deletions, inversions and translocations.

  • Programmable nucleases induce off-target mutations at sites that are highly homologous to their target sites. Measuring and reducing off-target effects of engineered nucleases is of great importance in research, biotechnology and medicine.

  • Programmable nickases derived from nucleases induce single-strand breaks (that is, 'nicks'), the repair of which can lead to precise genome editing. Paired nickases can induce genome editing as efficiently as nucleases but with a much higher specificity.

  • With programmable nucleases, artificial selection can be driven by desired genotypes a priori rather than by unpredictable phenotypes a posteriori.

Abstract

Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) system — enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.

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: Outcome of genome editing using programmable nucleases.
Figure 2: Structure of ZFNs.
Figure 3: Structure of TALENs.
Figure 4: Structure of RGENs.

Similar content being viewed by others

References

  1. Krueger, U. et al. Insights into effective RNAi gained from large-scale siRNA validation screening. Oligonucleotides 17, 237–250 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Jackson, A. L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotech. 21, 635–637 (2003).

    Article  CAS  Google Scholar 

  3. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 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). This paper describes targeted gene knockout in animals using engineered nucleases.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Cho, S. W., Kim, S., Kim, J. M. & Kim, J. S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nature Biotech. 31, 230–232 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR–Cas system. Nature Biotech. 31, 227–229 (2013).

    Article  CAS  Google Scholar 

  8. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nature Biotech. 31, 233–239 (2013).

    Article  CAS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Jinek, M. et al. RNA-programmed genome editing in human cells. Elife 2, e00471 (2013). References 5, 6, 9 and 10 show that RGENs can cleave chromosomal DNA and induce site-specific mutations efficiently in human cells.

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  14. Steentoft, C. et al. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines. Nature Methods 8, 977–982 (2011).

    Article  CAS  PubMed  Google Scholar 

  15. Kim, Y. et al. A library of TAL effector nucleases spanning the human genome. Nature Biotech. 31, 251–258 (2013). This paper presents a genome-scale library of TALENs that target human protein-coding genes.

    Article  CAS  Google Scholar 

  16. Kernstock, S. et al. Lysine methylation of VCP by a member of a novel human protein methyltransferase family. Nature Commun. 3, 1038 (2012).

    Article  CAS  Google Scholar 

  17. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  19. Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221 (2011). This study shows that ZFNs can be delivered in vivo and lead to gene correction in mice.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nature Biotech. 29, 731–734 (2011).

    Article  CAS  Google Scholar 

  21. Doyon, J. B. et al. Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells. Nature Cell Biol. 13, 331–337 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Urnov, F. D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005). This milestone paper in the field of genome editing presents efficient gene correction using ZFNs in human cell lines.

    Article  CAS  PubMed  Google Scholar 

  25. Chen, F. et al. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nature Methods 8, 753–755 (2011). This paper describes a convenient method to introduce user-defined variations in a gene of interest using ssODNs.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cui, X. et al. Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nature Biotech. 29, 64–67 (2011).

    Article  CAS  Google Scholar 

  27. Wefers, B. et al. Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. Proc. Natl. Acad. Sci. USA 110, 3782–3787 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  28. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Soldner, F. et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146, 318–331 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lee, H. J., Kim, E. & Kim, J. S. Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res. 20, 81–89 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lee, H. J., Kweon, J., Kim, E., Kim, S. & Kim, J. S. Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases. Genome Res. 22, 539–548 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Carlson, D. F. et al. Efficient TALEN-mediated gene knockout in livestock. Proc. Natl. Acad. Sci. USA 109, 17382–17387 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Xiao, A. et al. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res. 41, e141 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gupta, A. et al. Targeted chromosomal deletions and inversions in zebrafish. Genome Res. 23, 1008–1017 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Brunet, E. et al. Chromosomal translocations induced at specified loci in human stem cells. Proc. Natl. Acad. Sci. USA 106, 10620–10625 (2009). References 30, 31 and 35 show that programmable nucleases enable targeted chromosomal rearrangements in human cell lines to induce deletions, inversions, duplications and translocations.

    Article  PubMed  PubMed Central  Google Scholar 

  36. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Gaj, T., Gersbach, C. A. & Barbas, C. F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Segal, D. J. & Meckler, J. F. Genome engineering at the dawn of the golden age. Annu. Rev. Genom. Hum. Genet. 14, 135–158 (2013).

    Article  CAS  Google Scholar 

  39. Perez-Pinera, P., Ousterout, D. G. & Gersbach, C. A. Advances in targeted genome editing. Curr. Opin. Chem. Biol. 16, 268–277 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Bitinaite, J., Wah, D. A., Aggarwal, A. K. & Schildkraut, I. FokI dimerization is required for DNA cleavage. Proc. Natl. Acad. Sci. USA 95, 10570–10575 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  44. Szczepek, M. et al. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nature Biotech. 25, 786–793 (2007). References 43 and 44 introduce obligatory heterodimeric Fok I variants to enhance the specificity of ZFNs and TALENs.

    Article  CAS  Google Scholar 

  45. Guo, J., Gaj, T. & Barbas, C. F. 3rd. Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. J. Mol. Biol. 400, 96–107 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Doyon, Y. et al. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nature Methods 8, 74–79 (2011).

    Article  CAS  PubMed  Google Scholar 

  47. Tupler, R., Perini, G. & Green, M. R. Expressing the human genome. Nature 409, 832–833 (2001).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  49. Desjarlais, J. R. & Berg, J. M. Redesigning the DNA-binding specificity of a zinc finger protein: a data base-guided approach. Proteins 12, 101–104 (1992).

    Article  CAS  PubMed  Google Scholar 

  50. Rebar, E. J. & Pabo, C. O. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science 263, 671–673 (1994). This paper describes a powerful method to generate sequence-specific ZFPs using phage display and paved the way for the construction of ZFNs.

    Article  CAS  PubMed  Google Scholar 

  51. Pavletich, N. P. & Pabo, C. O. Zinc finger–DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 Å. Science 252, 809–817 (1991).

    Article  CAS  PubMed  Google Scholar 

  52. Segal, D. J. et al. Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins. Biochemistry 42, 2137–2148 (2003).

    Article  CAS  PubMed  Google Scholar 

  53. Bae, K. H. et al. Human zinc fingers as building blocks in the construction of artificial transcription factors. Nature Biotech. 21, 275–280 (2003).

    Article  CAS  Google Scholar 

  54. Kim, J. S., Lee, H. J. & Carroll, D. Genome editing with modularly assembled zinc-finger nucleases. Nature Methods 7, 91 (2010).

    Article  CAS  PubMed  Google Scholar 

  55. Ramirez, C. L. et al. Unexpected failure rates for modular assembly of engineered zinc fingers. Nature Methods 5, 374–375 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Cornu, T. I. et al. DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases. Mol. Ther. 16, 352–358 (2008).

    Article  CAS  PubMed  Google Scholar 

  57. Maeder, M. L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol. Cell 31, 294–301 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Sander, J. D. et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nature Methods 8, 67–69 (2011).

    Article  CAS  PubMed  Google Scholar 

  59. Gupta, A. et al. An optimized two-finger archive for ZFN-mediated gene targeting. Nature Methods 9, 588–590 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Segal, D. J., Dreier, B., Beerli, R. R. & Barbas, C. F. 3rd. Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences. Proc. Natl. Acad. Sci. USA 96, 2758–2763 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Kim, H. J., Lee, H. J., Kim, H., Cho, S. W. & Kim, J. S. Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res. 19, 1279–1288 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Sung, Y. H. et al. Knockout mice created by TALEN-mediated gene targeting. Nature Biotech. 31, 23–24 (2013).

    Article  CAS  Google Scholar 

  67. Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. Nature Biotech. 29, 695–696 (2011).

    Article  CAS  Google Scholar 

  68. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  70. Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nature Biotech. 29, 143–148 (2011). This paper shows the first successful gene knockout in human cells using an improved TALEN architecture.

    Article  CAS  Google Scholar 

  71. Li, L. et al. Characterization and DNA-binding specificities of Ralstonia TAL-like effectors. Mol. Plant 6, 1318–1330 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Doyle, E. L. et al. TAL effector specificity for base 0 of the DNA target is altered in a complex, effector- and assay-dependent manner by substitutions for the tryptophan in cryptic repeat −1. PLoS ONE 8, e82120 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J. & Stoddard, B. L. The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 335, 716–719 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Deng, D. et al. Structural basis for sequence-specific recognition of DNA by TAL effectors. Science 335, 720–723 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  76. Moscou, M. J. & Bogdanove, A. J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009). References 75 and 76 present the code of TALE–DNA interactions and paved the way for the construction of TALENs.

    Article  CAS  PubMed  Google Scholar 

  77. Heigwer, F. et al. E-TALEN: a web tool to design TALENs for genome engineering. Nucleic Acids Res. 41, e190 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Holkers, M. et al. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells. Nucleic Acids Res. 41, e63 (2013).

    Article  CAS  PubMed  Google Scholar 

  79. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nature Biotech. 30, 460–465 (2012).

    Article  CAS  Google Scholar 

  81. Schmid-Burgk, J. L., Schmidt, T., Kaiser, V., Honing, K. & Hornung, V. A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. Nature Biotech. 31, 76–81 (2013).

    Article  CAS  Google Scholar 

  82. Briggs, A. W. et al. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic Acids Res. 40, e117 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Ding, Q. et al. A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell 12, 238–251 (2013).

    Article  CAS  PubMed  Google Scholar 

  84. Kim, Y. K. et al. TALEN-based knockout library for human microRNAs. Nature Struct. Mol. Biol. 20, 1458–1464 (2013). This paper presents a genome-scale library of TALENs that target human miRNA sequences.

    Article  CAS  Google Scholar 

  85. Wang, Z. et al. An integrated chip for the high-throughput synthesis of transcription activator-like effectors. Angew. Chem. Int. Ed. Engl. 51, 8505–8508 (2012).

    Article  CAS  PubMed  Google Scholar 

  86. Lamb, B. M., Mercer, A. C. & Barbas, C. F. 3rd. Directed evolution of the TALE N-terminal domain for recognition of all 5′ bases. Nucleic Acids Res. 41, 9779–9785 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Sun, N., Liang, J., Abil, Z. & Zhao, H. Optimized TAL effector nucleases (TALENs) for use in treatment of sickle cell disease. Mol. Biosyst. 8, 1255–1263 (2012).

    Article  CAS  PubMed  Google Scholar 

  88. Bultmann, S. et al. Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers. Nucleic Acids Res. 40, 5368–5377 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Valton, J. et al. Overcoming transcription activator- like effector (TALE) DNA binding domain sensitivity to cytosine methylation. J. Biol. Chem. 287, 38427–38432 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Deng, D. et al. Recognition of methylated DNA by TAL effectors. Cell Res. 22, 1502–1504 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Li, J. F. et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotech. 31, 688–691 (2013).

    Article  CAS  Google Scholar 

  92. Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J. D. & Kamoun, S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nature Biotech. 31, 691–693 (2013).

    Article  CAS  Google Scholar 

  93. Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR–Cas system. Nature Biotech. 31, 686–688 (2013).

    Article  CAS  Google Scholar 

  94. Friedland, A. E. et al. Heritable genome editing in C. elegans via a CRISPR–Cas9 system. Nature Methods 10, 741–743 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Dickinson, D. J., Ward, J. D., Reiner, D. J. & Goldstein, B. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nature Methods 10, 1028–1034 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Cho, S. W., Lee, J., Carroll, D., Kim, J. S. & Lee, J. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9–sgRNA ribonucleoproteins. Genetics 195, 1177–1180 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Gratz, S. J. et al. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194, 1029–1035 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Li, D. et al. Heritable gene targeting in the mouse and rat using a CRISPR–Cas system. Nature Biotech. 31, 681–683 (2013).

    Article  CAS  Google Scholar 

  99. Sung, Y. H. et al. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res. 24, 125–131 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Shen, B. et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 23, 720–723 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Li, W., Teng, F., Li, T. & Zhou, Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR–Cas systems. Nature Biotech. 31, 684–686 (2013).

    Article  CAS  Google Scholar 

  102. Niu, Y. et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156, 836–843 (2014).

    Article  CAS  PubMed  Google Scholar 

  103. Ding, Q. et al. Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell 12, 393–394 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Makarova, K. S., Grishin, N. V., Shabalina, S. A., Wolf, Y. I. & Koonin, E. V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct 1, 7 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science http://dx.doi.org/10.1126/science.1247997 (2014).

  108. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotech. 31, 827–832 (2013).

    Article  CAS  Google Scholar 

  109. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotech. 31, 833–838 (2013). References 23, 36 and 109 present paired Cas9 nickases that produce two offset SSBs on opposite DNA strands to enhance the specificity of RNA-guided genome editing, which is a strategy originally implemented using paired ZFNickases (reference 142).

    Article  CAS  Google Scholar 

  110. Mojica, F. J., Diez-Villasenor, C., Garcia-Martinez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009).

    Article  CAS  PubMed  Google Scholar 

  111. Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Shah, S. A., Erdmann, S., Mojica, F. J. & Garrett, R. A. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol. 10, 891–899 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Hou, Z. et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl. Acad. Sci. USA 110, 15644–15649 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR–Cas systems. Nucleic Acids Res. 42, 2577–2590 (2014).

    Article  CAS  PubMed  Google Scholar 

  115. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). This paper shows that the Cas9 protein is guided by small RNAs to cleave DNA in a targeted manner in vitro , which paved the way for RNA-guided genome editing both in cells and in vivo.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Koike-Yusa, H., Li, Y., Tan, E.-P., Velasco-Herrera, M. D. C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nature Biotech. 32, 267–273 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Mussolino, C. et al. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 39, 9283–9293 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nature Biotech. 29, 816–823 (2011). References 117 and 119 demonstrate two different methods to identify off-target sites of programmable nucleases in the human genome.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  121. Cradick, T. J., Fine, E. J., Antico, C. J. & Bao, G. CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotech. 31, 839–843 (2013). References 36, 109 and 120–122 show that RGENs induce off-target mutations in human cells.

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Kim, Y., Kweon, J. & Kim, J. S. TALENs and ZFNs are associated with different mutation signatures. Nature Methods 10, 185 (2013).

    Article  PubMed  Google Scholar 

  125. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  126. Meng, X., Noyes, M. B., Zhu, L. J., Lawson, N. D. & Wolfe, S. A. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nature Biotech. 26, 695–701 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  128. Perez, E. E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nature Biotech. 26, 808–816 (2008). This study develops ZFNs that disrupt the chemokine (C-C motif) receptor 5 ( CCR5 ) gene in T cells to prevent HIV infection.

    Article  CAS  Google Scholar 

  129. Shalem, O. et al. Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    Article  CAS  PubMed  Google Scholar 

  130. Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR–Cas9 system. Science 343, 80–84 (2014). References 116, 129 and 130 describe genome-scale gene knockout screening in human and murine cell lines.

    Article  CAS  PubMed  Google Scholar 

  131. Gaj, T., Guo, J., Kato, Y., Sirk, S. J. & Barbas, C. F. 3rd. Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nature Methods 9, 805–807 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Chen, Z. et al. Receptor-mediated delivery of engineered nucleases for genome modification. Nucleic Acids Res. 41, e182 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Pruett-Miller, S. M., Reading, D. W., Porter, S. N. & Porteus, M. H. Attenuation of zinc finger nuclease toxicity by small-molecule regulation of protein levels. PLoS Genet. 5, e1000376 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Kim, J. M., Kim, D., Kim, S. & Kim, J. S. Genotyping with CRISPR–Cas-derived RNA-guided endonucleases. Nature Commun. 5, 3157 (2014).

    Article  CAS  Google Scholar 

  135. Li, T. et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res. 39, 6315–6325 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Yusa, K. et al. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells. Nature 478, 391–394 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. 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 http://dx.doi.org/10.1093/bioinformatics/btu048 (2014).

  138. Heigwer, F., Kerr, G. & Boutros, M. E-CRISP: fast CRISPR target site identification. Nature Methods 11, 122–123 (2014).

    Article  CAS  PubMed  Google Scholar 

  139. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR–Cas nuclease specificity using truncated guide RNAs. Nature Biotech. 32, 279–284 (2014).

    Article  CAS  Google Scholar 

  140. McConnell Smith, A. et al. Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease. Proc. Natl. Acad. Sci. USA 106, 5099–5104 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  141. Davis, L. & Maizels, N. DNA nicks promote efficient and safe targeted gene correction. PLoS ONE 6, e23981 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Kim, E. et al. Precision genome engineering with programmable DNA-nicking enzymes. Genome Res. 22, 1327–1333 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Wang, J. et al. Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme. Genome Res. 22, 1316–1326 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Ramirez, C. L. et al. Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects. Nucleic Acids Res. 40, 5560–5568 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Gabsalilow, L., Schierling, B., Friedhoff, P., Pingoud, A. & Wende, W. Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats. Nucleic Acids Res. 41, e83 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Doyon, Y. et al. Transient cold shock enhances zinc-finger nuclease-mediated gene disruption. Nature Methods 7, 459–460 (2010).

    Article  CAS  PubMed  Google Scholar 

  148. Ramakrishna, S., Kim, Y. H. & Kim, H. Stability of zinc finger nuclease protein is enhanced by the proteasome inhibitor MG132. PLoS ONE 8, e54282 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. Herrmann, F. et al. p53 gene repair with zinc finger nucleases optimised by yeast 1-hybrid and validated by Solexa sequencing. PLoS ONE 6, e20913 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  150. Certo, M. T. et al. Coupling endonucleases with DNA end-processing enzymes to drive gene disruption. Nature Methods 9, 973–975 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. Mashimo, T. et al. Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes. Sci. Rep. 3, 1253 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Frank, S., Skryabin, B. V. & Greber, B. A modified TALEN-based system for robust generation of knock-out human pluripotent stem cell lines and disease models. BMC Genomics 14, 773 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  153. Kim, H., Um, E., Cho, S. R., Jung, C. & Kim, J. S. Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nature Methods 8, 941–943 (2011).

    Article  CAS  PubMed  Google Scholar 

  154. Kim, H. et al. Magnetic separation and antibiotics selection enable enrichment of cells with ZFN/TALEN-induced mutations. PLoS ONE 8, e56476 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  155. Ramakrishna, S. et al. Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nature Commun. 5, 3414 (2014).

    Article  CAS  Google Scholar 

  156. Certo, M. T. et al. Tracking genome engineering outcome at individual DNA breakpoints. Nature Methods 8, 671–676 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  157. Beerli, R. R., Dreier, B. & Barbas, C. F. 3rd. Positive and negative regulation of endogenous genes by designed transcription factors. Proc. Natl. Acad. Sci. USA 97, 1495–1500 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  158. Mendenhall, E. M. et al. Locus-specific editing of histone modifications at endogenous enhancers. Nature Biotech. 31, 1133–1136 (2013).

    Article  CAS  Google Scholar 

  159. Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  160. Maeder, M. L. et al. Targeted DNA demethylation and activation of endogenous genes using programmable TALE–TET1 fusion proteins. Nature Biotech. 31, 1137–1142 (2013).

    Article  CAS  Google Scholar 

  161. Miyanari, Y., Ziegler-Birling, C. & Torres-Padilla, M. E. Live visualization of chromatin dynamics with fluorescent TALEs. Nature Struct. Mol. Biol. 20, 1321–1324 (2013).

    Article  CAS  Google Scholar 

  162. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  163. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  164. Sigoillot, F. D. & King, R. W. Vigilance and validation: keys to success in RNAi screening. ACS Chem. Biol. 6, 47–60 (2011).

    Article  CAS  PubMed  Google Scholar 

  165. Dawkins, R. The Blind Watchmaker: Why the Evidence of Evolution Reveals a Universe without Design (Norton & Company, 1986).

  166. Zeevi, V., Tovkach, A. & Tzfira, T. Increasing cloning possibilities using artificial zinc finger nucleases. Proc. Natl Acad. Sci. USA 105, 12785–12790 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  167. Smidler, A. L., Terenzi, O., Soichot, J., Levashina, E. A. & Marois, E. Targeted mutagenesis in the malaria mosquito using TALE nucleases. PLoS ONE 8, e74511 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  168. Hauschild, J. et al. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 108, 12013–12017 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  169. Hu, R., Wallace, J., Dahlem, T. J., Grunwald, D. J. & O'Connell, R. M. Targeting human microRNA genes using engineered Tal-effector nucleases (TALENs). PLoS ONE 8, e63074 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  170. Gutschner, T., Baas, M. & Diederichs, S. Noncoding RNA gene silencing through genomic integration of RNA destabilizing elements using zinc finger nucleases. Genome Res. 21, 1944–1954 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  171. Zou, J., Mali, P., Huang, X., Dowey, S. N. & Cheng, L. Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease. Blood 118, 4599–4608 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  172. Simsek, D. et al. DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation. PLoS Genet. 7, e1002080 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  173. Carbery, I. D. et al. Targeted genome modification in mice using zinc-finger nucleases. Genetics 186, 451–459 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  174. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  175. Geurts, A. M. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  176. Doyon, Y. et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nature Biotech. 26, 702–708 (2008).

    Article  CAS  Google Scholar 

  177. Zou, J. et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 5, 97–110 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  179. Townsend, J. A. et al. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459, 442–445 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  180. Li, T., Liu, B., Spalding, M. H., Weeks, D. P. & Yang, B. High-efficiency TALEN-based gene editing produces disease-resistant rice. Nature Biotech. 30, 390–392 (2012).

    Article  CAS  Google Scholar 

  181. Yu, S. et al. Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle. Cell Res. 21, 1638–1640 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  182. Santiago, Y. et al. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105, 5809–5814 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  184. Maier, D. A. et al. Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5. Hum. Gene Ther. 24, 245–258 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  185. Osborn, M. J. et al. TALEN-based gene correction for epidermolysis bullosa. Mol. Ther. 21, 1151–1159 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  186. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  187. 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).

    Article  CAS  PubMed  Google Scholar 

  188. Sebastiano, V. et al. In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases. Stem Cells 29, 1717–1726 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  189. Zou, J. et al. Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting. Blood 117, 5561–5572 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  190. Jiang, J. et al. Translating dosage compensation to trisomy 21. Nature 500, 296–300 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  191. Carroll, D. Genome engineering with zinc-finger nucleases. Genetics 188, 773–782 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  192. Kim, S., Lee, M. J., Kim, H., Kang, M. & Kim, J. S. Preassembled zinc-finger arrays for rapid construction of ZFNs. Nature Methods 8, 7 (2011).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Research Foundation of Korea (2013-000718 to J.S.K. and 2011-0013568 to H.K.), Health Technology Research and Development Project by the Korean Ministry of Health and Welfare (H10C1740 to H.K.), and Converging Research Center Program funded by the Korean Ministry of Science, ICT and Future Planning (2013K000275 to H.K.). The authors thank three anonymous referees for comments and all the researchers, including our collaborators and competitors, who have contributed to the development of programmable nucleases in the past 20 years and regret the need to omit many relevant papers owing to the page limitation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jin-Soo Kim.

Ethics declarations

Competing interests

The authors hold stocks in ToolGen, Inc. mentioned in this article.

Related links

PowerPoint slides

Glossary

Homologous recombination

A genetic recombination process in which two similar DNA strands exchange nucleotide sequences.

Non-homologous end-joining

(NHEJ). A repair pathway for DNA double-strand breaks (DSBs) through direct ligation of the break ends without using a homologous template. This error-prone process often causes small insertions and deletions at the DSB site.

Zinc-finger nucleases

(ZFNs). Programmable nucleases composed of the FokI catalytic domain and zinc-finger DNA-binding domains.

Transcription activator-like effector nucleases

(TALENs). Programmable nucleases composed of the FokI catalytic domain and TALE proteins.

RNA-guided engineered nucleases

(RGENs). Programmable nucleases composed of the Cas9 protein and a guide RNA.

Clustered regularly interspaced short palindromic repeat

(CRISPR). A genomic locus in bacteria or archaea where protospacers and direct repeat sequences are arrayed in tandem. It is associated with adaptive immunity against invading phages and plasmids.

Homology-directed repair

(HDR). Template-dependent repair of DNA strand breaks using homologous DNA sequences such as double-strand donor DNA or single-strand oligonucleotides. HDR of programmable nuclease-induced strand breaks leads to precise genome editing, including targeted gene insertion, correction and point mutagenesis.

Insertions and deletions

(Indels). Small insertions or deletions of DNA sequences relative to a reference sequence.

FokI

A type IIS restriction enzyme found in Flavobacterium okeanokoites that is composed of a separable DNA-binding domain and a nuclease domain that is used to construct ZFNs and TALENs.

Transcription activator-like effectors

(TALEs). DNA-binding proteins with a modular structure derived from Xanthomonas spp. (a plant pathogen). Each module is composed of ~34 amino acids and recognizes a single nucleotide. The base specificity is determined by the amino acids at positions 12 and 13 (known as repeat variable diresidues) in each module.

Protospacers

DNA sequences of 26–72 bp that are initially derived from invading phages and plasmids and that are embedded in the CRISPR loci in bacteria or archaea.

CRISPR RNA

(crRNA). A small RNA transcribed from the CRISPR loci that determines the target-sequence specificity of Cas9 RNA-guided endonulceases.

CRISPR-associated protein 9

(Cas9). A protein derived from bacteria such as Streptococcus pyogenes. The Cas9 protein forms an active DNA endonuclease when complexed with guide RNAs.

Protospacer adjacent motif

(PAM). A short (2–5-bp) nucleotide motif adjacent to protospacers that is recognized by Cas9.

Single-chain guide RNA

(sgRNA). A small, single-chain guide RNA that is created by the fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).

Nickases

Enzymes that generate DNA single-strand breaks (that is, 'nicks').

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, H., Kim, JS. A guide to genome engineering with programmable nucleases. Nat Rev Genet 15, 321–334 (2014). https://doi.org/10.1038/nrg3686

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrg3686

This article is cited by

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