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:

Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation

Key Points

  • Beyond gene editing, the CRISPR–Cas9 technology offers a versatile sequence-specific gene regulation 'toolset', by utilizing the nuclease-deficient dCas9, which was designed not to cleave DNA but to precisely and specifically bind DNA when guided by a single guide RNA (sgRNA).

  • In CRISPR interference (CRISPRi), dCas9 is targeted to block transcription and thereby silence genes. Improvements include the fusion of dCas9 to transcriptional repressors for increased repression efficiency.

  • CRISPR activation (CRISPRa) uses dCas9 fusion proteins to recruit transcription activators for targeted gene activation. The use of enhanced dCas9 activation systems allows recruitment of multiple activators with one sgRNA.

  • dCas9 can direct epigenetic modifications at specific genomic locations, through the use of dCas9 fusion proteins that recruit epigenetic modifiers that can alter epigenetic marks at enhancers and other regulatory elements.

  • Rational engineering of the sgRNA molecule by the addition of aptamers allows for the recruitment of transcription repressors or activators, and the simultaneous activation of one gene target and repression of another gene target, using one dCas9 protein.

  • CRISPRi and CRISPRa are highly gene-specific.

  • Applications of dCas9 currently include genome-wide loss-of-function or gain-of function screens, inducible and reversible gene regulation and cell fate engineering.

Abstract

The bacterial CRISPR–Cas9 system has emerged as a multifunctional platform for sequence-specific regulation of gene expression. This Review describes the development of technologies based on nuclease-deactivated Cas9, termed dCas9, for RNA-guided genomic transcription regulation, both by repression through CRISPR interference (CRISPRi) and by activation through CRISPR activation (CRISPRa). We highlight different uses in diverse organisms, including bacterial and eukaryotic cells, and summarize current applications of harnessing CRISPR–dCas9 for multiplexed, inducible gene regulation, genome-wide screens and cell fate engineering. We also provide a perspective on future developments of the technology and its applications in biomedical research and clinical studies.

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: Gene editing versus gene regulation using Streptococcus pyogenes Cas9 and dCas9.
Figure 2: CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) for transcription repression and activation.
Figure 3: Simultaneous transcription activation and repression.
Figure 4: Applications of the CRISPR–dCas9 technology.

Similar content being viewed by others

References

  1. Mohr, S. E., Smith, J. A., Shamu, C. E., Neumuller, R. A. & Perrimon, N. RNAi screening comes of age: improved techniques and complementary approaches. Nat. Rev. Mol. Cell Biol. 15, 591–600 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  3. 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 

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

    PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  9. Kabadi, A. M. & Gersbach, C. A. Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression. Methods 69, 188–197 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 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  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  13. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). This paper shows that Cas9, when paired with a tracrRNA–crRNA complex or a chimeric sgRNA, can recognize and cut specific DNA sequences.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 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  PubMed  PubMed Central  Google Scholar 

  15. Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013). This paper describes the nuclease-deficient Cas9 (dCas9) and its use as an RNA-guided DNA-binding platform for gene repression in bacteria and mammalian cells.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Makarova, K. S. et al. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol. 13, 1–15 (2015).

    Google Scholar 

  17. 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 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013). Along with references 21 and 22, this paper demonstrates the use of the nuclease Cas9 for genome editing in mammalian cells.

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 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 

  26. Doudna, J. A. & Charpentier, E. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014).

    PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Sander, J. D. & Joung, J. K. CRISPR–Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Larson, M. H. et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8, 2180–2196 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 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 

  32. Ma, H. et al. Multicolor CRISPR labeling of chromosomal loci in human cells. Proc. Natl Acad. Sci. USA 112, 3002–3007 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Fujita, T. & Fujii, H. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun. 439, 132–136 (2013).

    CAS  PubMed  Google Scholar 

  34. Fujita, T. et al. Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Sci. Rep. 3, 3171 (2013).

    PubMed  PubMed Central  Google Scholar 

  35. Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014). This paper demonstrates an optimized dCas9 system fused with a tandem peptide array (SunTag) for enhanced transcription activation.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Ji, W. et al. Specific gene repression by CRISPRi system transferred through bacterial conjugation. ACS Synth. Biol. 3, 929–931 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Lawhorn, I. E., Ferreira, J. P. & Wang, C. L. Evaluation of sgRNA target sites for CRISPR-mediated repression of TP53. PLoS ONE 9, e113232 (2014).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014). This paper describes the use of dCas9 fusion proteins for gain-of-function and loss-of-function screens.

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Farzadfard, F., Perli, S. D. & Lu, T. K. Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS Synth. Biol. 2, 604–613 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155–e155 (2014).

    PubMed  PubMed Central  Google Scholar 

  46. 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). This paper, together with references 38 and 42–45, describes the use of dCas9 fused with the repressor KRAB or the activator VP64 for manipulating gene expression in mammalian and yeast cells.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015). This paper demonstrates the use of a tripartite system (VPR) for efficient transcription activation.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 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 

  49. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2014). This paper describes the use of an engineered activator system (SAM) for efficient transcription activation and for genome-wide activation screening.

    PubMed  PubMed Central  Google Scholar 

  50. Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nat. Methods 12, 401–403 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Yeom, Y. I. et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894 (1996).

    CAS  PubMed  Google Scholar 

  52. Thakore, P. I. et al. Highly specific epigenome editing by CRISPR–Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015). Along with reference 50, this paper describes the fusion of dCas9 with a histone-modifying enzyme for targeted epigenetic modifications.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Keung, A. J., Bashor, C. J., Kiriakov, S., Collins, J. J. & Khalil, A. S. Using targeted chromatin regulators to engineer combinatorial and spatial transcriptional regulation. Cell 158, 110–120 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Zalatan, J. G. et al. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339–350 (2015). This paper describes the development of scRNAs with dCas9 for multiplexed and parallel transcription activation and repression in mammalian cells.

    CAS  PubMed  Google Scholar 

  56. Briner, A. E. et al. Guide RNA functional modules direct cas9 activity and orthogonality. Mol. Cell 56, 333–339 (2014).

    CAS  PubMed  Google Scholar 

  57. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Kuscu, C., Arslan, S., Singh, R., Thorpe, J. & Adli, M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat. Biotechnol. 32, 677–683 (2014).

    CAS  PubMed  Google Scholar 

  62. O'Geen, H., Henry, I. M., Bhakta, M. S., Meckler, J. F. & Segal, D. J. A genome-wide analysis of Cas9 binding specificity using ChIP–seq and targeted sequence capture. Nucleic Acids Res. 43, 3389–3404 (2015).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Polstein, L. R. et al. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators. Genome Res. 25, 1158–1169 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Bassik, M. C. et al. A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility. Cell 152, 909–922 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera Mdel, C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267–273 (2014).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  72. 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).

    CAS  PubMed  Google Scholar 

  73. Zhou, Y. et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509, 487–491 (2014).

    CAS  PubMed  Google Scholar 

  74. Kampmann, M. et al. Next-generation libraries for robust RNA interference-based genome-wide screens. Proc. Natl Acad. Sci. USA 112, E3384–E3391 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR–Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Nihongaki, Y., Yamamoto, S., Kawano, F., Suzuki, H. & Sato, M. CRISPR–Cas9-based photoactivatable transcription system. Chem. Biol. 22, 169–174 (2015).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  78. Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR–Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).

    CAS  PubMed  Google Scholar 

  79. Hu, J. et al. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors. Nucleic Acids Res. 42, 4375–4390 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Balboa, D. et al. Conditionally stabilized dCas9 activator for controlling gene expression in human cell reprogramming and differentiation. Stem Cell Rep. 5, 448–594 (2015).

    CAS  Google Scholar 

  81. Chakraborty, S. et al. A CRISPR/Cas9-Based system for reprogramming cell lineage specification. Stem Cell Rep. 3, 940–947 (2014).

    CAS  Google Scholar 

  82. Kearns, N. A. et al. Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. Development 141, 219–223 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–384 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Platt, R. J. et al. CRISPR–Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  87. 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).

    CAS  PubMed  Google Scholar 

  88. Maurano, M. T. et al. Systematic localization of common disease-associated variation in regulatory DNA. Science 337, 1190–1195 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank the members of the Qi lab for advice and helpful discussions. L.S.Q. acknowledges support from the U.S. National Institutes of Health (NIH) Office of the Director (OD) and National Institute of Dental & Craniofacial Research (NIDCR). A.A.D. acknowledges support through the Milton Safenowitz Post Doctoral Fellowship for ALS Research. This work was supported by NIH R01 DA036858 (to W.A.L. and L.S.Q.), the Howard Hughes Medical Institute (grant to W.A.L.) and NIH DP5 OD017887 (to A.A.D. and L.S.Q.).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Wendell A. Lim or Lei S. Qi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

PowerPoint slides

Glossary

CRISPR–Cas

(Clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins). CRISPR are bacterial DNA loci containing short repeat segments that match foreign DNA elements. Together with Cas proteins, they form an adaptive immune system in bacteria and archaea, which can acquire sequence segments from foreign DNA and use these sequences to recognize and destroy the foreign target DNA.

Type II CRISPR system

CRISPR–Cas system that encodes cas9, cas1 and cas2 within the CRISPR–cas loci, in addition to a tracrRNA, which is partially complementary to repeats in the CRISPR array.

Single guide RNA

(sgRNA). A synthetic RNA chimera containing a hairpin that links the transactivating CRISPR RNA (tracrRNA) to the crRNA and functions similarly to the native crRNA–tracrRNA duplex, directing Cas9 to a specific genomic locus.

Protospacer-adjacent motif

(PAM). A short sequence in the target DNA (not in the guide RNA) that is necessary for the successful targeting of Cas9. The PAM sequence varies between bacterial species. In Streptococcus pyogenes, it is NGG (which is more effective) or NAG (less effective).

Transactivating CRISPR RNA

(tracrRNA). A small RNA encoded upstream of the CRISPR locus in type II CRISPR systems, with a 24-nucleotide sequence complementary to repeats of the CRISPR RNA (crRNA) precursor transcripts. Essential for the processing of pre-crRNA to mature crRNA.

CRISPR RNA

(crRNA). Small RNAs transcribed from the protospacer within the CRISPR array. Together with the transactivating crRNA (tracrRNA), crRNA guides Cas9 to a specific genomic locus.

Krüppel-associated box

(KRAB). A conserved domain of a transcription repressor that can be fused to DNA-binding proteins for targeted transcription repression.

mSin3 interaction domains

Interaction domains that are present on multiple transcriptional repressor proteins.

VP64

A transcription activator composed of four tandem copies of the herpes simplex virus VP16 activation domain connected by Gly-Ser linkers. VP64 is often fused to DNA-binding proteins for targeted transcription activation.

p65 activation domain

(p65AD). The principal transactivation domain of the 65 kDa polypeptide of the nuclear form of the NF-κB transcription factor.

Single-chain variable fragment

(scFV). A fusion protein in which the epitope-binding regions of the heavy and light chains of an antibody are connected by a short linker peptide and are expressed in soluble form in cells.

Mediator complex

A multi-subunit complex that is required for the transcription of most RNA polymerase II transcripts.

RNA aptamers

RNA molecules that have high affinity and specificity for target molecules.

Optogenetics

A technique that utilizes optics for achieving spatiotemporal gene regulation of cells in living tissues.

CRY–CIB heterodimerizing domains

A light-inducible protein interaction between the blue light-sensitive cryptochrome 2 protein (CRY2) and its interacting partner CIB1 from Arabidopsis thaliana.

Direct lineage reprogramming

The conversion of fully differentiated cells of a certain type into another cell type, while bypassing the intermediate pluripotent state.

Adeno-associated virus (AAV) vectors

Viral vectors with small packaging capacity, commonly used in gene therapy, which can infect both dividing and non-dividing cells and do not integrate into the host genome. AAV vectors have been approved for clinical use.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dominguez, A., Lim, W. & Qi, L. Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol 17, 5–15 (2016). https://doi.org/10.1038/nrm.2015.2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrm.2015.2

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