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.

CRISPR-Cas systems for editing, regulating and targeting genomes

Abstract

Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Nuclease-induced genome editing.
Figure 2: Overview of various Cas9-based applications.
Figure 3: Naturally occurring and engineered CRISPR-Cas systems.
Figure 4: Cas9-based systems for altering gene sequence or expression.
Figure 5: Sequence limitations on the targeting range of guide RNAs.

References

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wiedenheft, B., Sternberg, S.H. & Doudna, J.A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012).

    CAS  Article  PubMed  Google Scholar 

  3. Fineran, P.C. & Charpentier, E. Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new information. Virology 434, 202–209 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 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  Article  PubMed  PubMed Central  Google Scholar 

  10. Esvelt, K.M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116–1121 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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 

  12. 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 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Jinek, M. et al. RNA-programmed genome editing in human cells. Elife 2, e00471 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. DiCarlo, J.E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  21. Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686–688 (2013).

    Article  CAS  PubMed  Google Scholar 

  22. Xie, K. & Yang, Y. RNA-guided genome editing in plants using a CRISPR-Cas system. Mol. Plant 6, 1975–1983 (2013).

    Article  CAS  PubMed  Google Scholar 

  23. Jiang, W. et al. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. 41, e188 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 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  Article  PubMed  PubMed Central  Google Scholar 

  25. 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 

  26. Li, D. et al. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat. Biotechnol. 31, 681–683 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Yang, D. et al. Effective gene targeting in rabbits using RNA-guided Cas9 nucleases. J. Mol. Cell Biol. 10.1093/jmcb/mjt047 (8 January 2014).

  28. Nakayama, T., et al. Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis. Genesis 51, 835–843 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yu, Z. et al. Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics 195, 289–291 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Bassett, A.R., Tibbit, C., Ponting, C.P. & Liu, J.L. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Reports 4, 220–228 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Wang, Y. et al. The CRISPR/Cas system mediates efficient genome engineering in Bombyx mori. Cell Res. 23, 1414–1416 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  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. Upadhyay, S.K., Kumar, J., Alok, A. & Tuli, R. RNA-guided genome editing for target gene mutations in wheat. G3 (Bethesda) 3, 2233–2238 (2013).

    Article  CAS  Google Scholar 

  35. Horii, T. et al. Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system. PeerJ 1, e230 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  37. Jao, L.E., Wente, S.R. & Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl. Acad. Sci. USA 110, 13904–13909 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  38. 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).

    Article  CAS  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  40. 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. Nat. Biotechnol. 32, 267–273 (2014).

    Article  CAS  PubMed  Google Scholar 

  41. Haft, D.H., Selengut, J., Mongodin, E.F. & Nelson, K.E. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol. 1, e60 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 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 

  43. 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 

  44. 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 

  45. 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 

  46. 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 

  47. 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 

  48. Fu, Y., Sander, J.D., Reyon, D., Cascio, V.M. & Joung, J.K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 10.1038/nbt.2808 (26 January 2014).

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. Semenova, E. et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl. Acad. Sci. USA 108, 10098–10103 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Wiedenheft, B. et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl. Acad. Sci. USA 108, 10092–10097 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 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 

  57. Kuzminov, A. Single-strand interruptions in replicating chromosomes cause double-strand breaks. Proc. Natl. Acad. Sci. USA 98, 8241–8246 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Cortes-Ledesma, F. & Aguilera, A. Double-strand breaks arising by replication through a nick are repaired by cohesin-dependent sister-chromatid exchange. EMBO Rep. 7, 919–926 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 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 

  60. Hwang, W.Y. et al. Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLoS ONE 8, e68708 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. 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 

  62. Lo, T.W. et al. Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. Genetics 195, 331–348 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Waaijers, S. et al. CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans. Genetics 195, 1187–1191 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Tzur, Y.B. et al. Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR-Cas9 system. Genetics 195, 1181–1185 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Katic, I. & Grosshans, H. Targeted heritable mutation and gene conversion by Cas9-CRISPR in Caenorhabditis elegans. Genetics 195, 1173–1176 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Chiu, H., Schwartz, H.T., Antoshechkin, I. & Sternberg, P.W. Transgene-free genome editing in Caenorhabditis elegans using CRISPR-Cas. Genetics 195, 1167–1171 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Cho, S.W., Lee, J., Carroll, D. & Kim, J.S. 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 

  68. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Bikard, D. et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41, 7429–7437 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Maeder, M.L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. 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 

  74. Cheng, A.W. et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163–1171 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Maeder, M.L. et al. Robust, synergistic regulation of human gene expression using TALE activators. Nat. Methods 10, 243–245 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Perez-Pinera, P. et al. Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nat. Methods 10, 239–242 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  78. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Chylinski, K., Le Rhun, A. & Charpentier, E. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol. 10, 726–737 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Ebina, H., Misawa, N., Kanemura, Y. & Koyanagi, Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep 3, 2510 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  85. Chang, N. et al. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res. 23, 465–472 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Silva, G. et al. Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Curr. Gene Ther. 11, 11–27 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. 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  PubMed  Google Scholar 

  89. 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 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  92. 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 

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

    Article  CAS  PubMed  Google Scholar 

  94. Gonzalez, B. et al. Modular system for the construction of zinc-finger libraries and proteins. Nat. Protoc. 5, 791–810 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Wright, D.A. et al. Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat. Protoc. 1, 1637–1652 (2006).

    Article  PubMed  Google Scholar 

  96. Carroll, D., Morton, J.J., Beumer, K.J. & Segal, D.J. Design, construction and in vitro testing of zinc finger nucleases. Nat. Protoc. 1, 1329–1341 (2006).

    Article  CAS  PubMed  Google Scholar 

  97. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30, 460–465 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Boch, J. & Bonas, U. Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu. Rev. Phytopathol. 48, 419–436 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  101. 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 

Download references

Acknowledgements

J.K.J. is grateful for support from the US National Institutes of Health (NIH) (grants DP1 GM105378 and R01 GM088040), the Defense Advanced Research Projects Agency (grant W911NF-11-2-0056) and The Jim and Ann Orr Massachusetts General Hospital Research Scholar Award. This material is based upon work supported fully or in part by the US Army Research Laboratory and the US Army Research Office under grant number W911NF-11-2-0056. The authors apologize to colleagues whose studies were not cited due to length and reference constraints.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to J Keith Joung.

Ethics declarations

Competing interests

J.K.J. has financial interests in Editas Medicine and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. J.K.J. and J.D.S. are consultants for Editas Medicine.

Supplementary information

Supplementary Table 1

Comparison of dgRNAs and sgRNAs used in various published studies. (PDF 191 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sander, J., Joung, J. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32, 347–355 (2014). https://doi.org/10.1038/nbt.2842

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2842

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