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:

Applications of CRISPR–Cas systems in neuroscience

Nature Reviews Neuroscience volume 17, pages 36–44 (2016)Cite this article

Subjects

Key Points

  • Enzymes from the CRISPR–Cas (clustered regularly interspaced short palindromic repeat–CRISPR-associated protein) systems are powerful tools for genome editing in neuroscience research.

  • The DNA-targeting specificity of Cas proteins is RNA-guided, and target-specific RNA guides can be easily constructed to achieve single and multiplex gene editing in almost any cell type and organism. This enables precise genetic modifications in animal and cellular models on a large scale, and makes gene editing possible in non-traditional models.

  • Enzymatically inactive versions of Cas9 (known as dead Cas9) can be coupled to different functional domains to achieve targeted transcriptional control and epigenetic modification.

  • Cas proteins and RNA guides can be delivered into the brain for genome editing to enable precise genetic dissection of neuronal circuits and modelling of neurological disorders.

  • Cas-mediated genome editing in combination with induced pluripotent stem cells from human donors enables the study of complex neurological disorders on a large scale in vitro.

Abstract

Genome-editing tools, and in particular those based on CRISPR–Cas (clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein) systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in almost any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal models and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR–Cas systems has the potential to advance both basic and translational neuroscience research.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Genome-editing applications of CRISPR–Cas9.
Figure 2: Using Cas9 to generate genetically modified rodents and for in vivo genome editing.
Figure 3: In vitro applications of Cas9 in human iPSCs.

Similar content being viewed by others

References

  1. Lewis, E. B. & Bacher, F. Methods for feeding ethyl methane sulfonate (EMS) to Drosophila males. Drosoph. Inf. Serv. 43, 193–194 (1968).

    Google Scholar 

  2. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).

    Article  CAS  PubMed  Google Scholar 

  3. St Johnston, D. The art and design of genetic screens: Drosophila melanogaster. Nat. Rev. Genet. 3, 176–188 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Jorgensen, E. M. & Mango, S. E. The art and design of genetic screens: Caenorhabditis elegans. Nat. Rev. Genet. 3, 356–369 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Patton, E. E. & Zon, L. I. The art and design of genetic screens: zebrafish. Nat. Rev. Genet. 2, 956–966 (2001).

    Article  CAS  PubMed  Google Scholar 

  6. Thomas, K. R. & Capecchi, M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512 (1987).

    Article  CAS  PubMed  Google Scholar 

  7. Oddo, S. et al. Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Aβ and synaptic dysfunction. Neuron 39, 409–421 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Jeon, I. et al. Neuronal properties, in vivo effects, and pathology of a Huntington's disease patient-derived induced pluripotent stem cells. Stem Cells 30, 2054–2062 (2012).

    Article  CAS  PubMed  Google Scholar 

  9. Yagi, T. et al. Modeling familial Alzheimer's disease with induced pluripotent stem cells. Hum. Mol. Genet. 20, 4530–4539 (2011).

    Article  CAS  PubMed  Google Scholar 

  10. Ryan, S. D. et al. Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription. Cell 155, 1351–1364 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Soldner, F. et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146, 318–331 (2011). References 10 and 11 combine ZFN-mediated genome-editing and human-stem-cell technologies for studying neurological disorders in vitro.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Pak, C. et al. Human neuropsychiatric disease modeling using conditional deletion reveals synaptic transmission defects caused by heterozygous mutations in NRXN1. Cell Stem Cell 17, 316–328 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. Bibikova, M. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell. Biol. 21, 289–297 (2001). This early study shows the use of ZFNs in Xenopus laevis for stimulating HR.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  16. 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 

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

    Article  CAS  PubMed  Google Scholar 

  18. 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 

  19. Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011). This paper uses an improved TALEN architecture to introduce gene knockouts in human cells.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  21. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007). This work provides the first experimental demonstration of the adaptive immune function of the CRISPR–Cas9 system in bacteria.

    Article  CAS  PubMed  Google Scholar 

  22. 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 

  23. Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010). This paper demonstrates that Cas9 facilitates RNA-guided DNA cleavage in bacteria.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013). References 24 and 25 describe the successful harnessing of the CRISPR–Cas9 system for editing the mammalian genome in cell lines.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Makarova, K. S. & Koonin, E. V. Annotation and classification of CRISPR–Cas systems. Methods Mol. Biol. 1311, 47–75 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Beerli, R. R., Segal, D. J., Dreier, B. & Barbas, C. F. Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc. Natl Acad. Sci. USA 95, 14628–14633 (1998).

    Article  CAS  PubMed  Google Scholar 

  29. 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 

  30. 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 

  31. 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 

  32. 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 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015).

    Article  CAS  PubMed  Google Scholar 

  35. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 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 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Straub, C., Granger, A. J., Saulnier, J. L. & Sabatini, B. L. CRISPR/Cas9-mediated gene knock-down in post-mitotic neurons. PLoS ONE 9, e105584 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Incontro, S., Asensio, C. S., Edwards, R. H. & Nicoll, R. A. Efficient, complete deletion of synaptic proteins using CRISPR. Neuron 83, 1051–1057 (2014). This paper reports the delivery of Cas9 and guide RNAs in organotypic brain-slice cultures and the disruption NMDA receptor and AMPA receptor subunits.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR–Cas9. Nat. Biotechnol. 33, 102–106 (2015). This paper demonstrates the delivery of Cas9 and guide RNAs into the mouse brain using AAV, and single and multiplex gene editing in vivo . It also shows a purification method of genetically tagged Cas9-targeted cell nuclei for DNA and RNA sequencing.

    Article  CAS  PubMed  Google Scholar 

  42. Shen, Z. et al. Conditional knockouts generated by engineered CRISPR–Cas9 endonuclease reveal the roles of coronin in C. elegans neural development. Dev. Cell 30, 625–636 (2014). This paper describes a conditional-knockout strategy using Cas9 in Caenorhabditis elegans for studying gene function in neural development.

    Article  CAS  PubMed  Google Scholar 

  43. Shah, A. N., Davey, C. F., Whitebirch, A. C., Miller, A. C. & Moens, C. B. Rapid reverse genetic screening using CRISPR in zebrafish. Nat. Methods 12, 535–540 (2015). This paper represents a useful application of Cas9 for studying neurodevelopmental processes on a large scale in zebrafish.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Auer, T. O., Duroure, K., De Cian, A., Concordet, J. P. & Del Bene, F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res. 24, 142–153 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 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  Google Scholar 

  46. Zuckermann, M. et al. Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling. Nat. Commun. 6, 7391 (2015). This paper describes methods for delivering Cas9 and guide RNA into the brains of newborn mice and embryos. By targeting multiple tumour-suppressor genes, the development of the medulla and glioblastoma was induced.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Fishman-Lobell, J. & Haber, J. E. Removal of nonhomologous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene RAD1. Science 258, 480–484 (1992).

    Article  CAS  PubMed  Google Scholar 

  50. Fishman-Lobell, J., Rudin, N. & Haber, J. E. Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated. Mol. Cell. Biol. 12, 1292–1303 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Liang, F., Han, M., Romanienko, P. J. & Jasin, M. Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc. Natl Acad. Sci. USA 95, 5172–5177 (1998).

    Article  CAS  PubMed  Google Scholar 

  52. Johnson, R. D., Liu, N. & Jasin, M. Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination. Nature 401, 397–399 (1999).

    CAS  PubMed  Google Scholar 

  53. 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 

  54. 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 

  55. Rouet, P., Smih, F. & Jasin, M. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc. Natl Acad. Sci. USA 91, 6064–6068 (1994).

    Article  CAS  PubMed  Google Scholar 

  56. 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). This paper, along with reference 37, characterizes Cas9-mediated DNA cleavage in vitro.

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Brenowitz, E. A. & Zakon, H. H. Emerging from the bottleneck: benefits of the comparative approach to modern neuroscience. Trends Neurosci. 38, 273–278 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  PubMed  Google Scholar 

  60. Hommel, J. D., Sears, R. M., Georgescu, D., Simmons, D. L. & DiLeone, R. J. Local gene knockdown in the brain using viral-mediated RNA interference. Nat. Med. 9, 1539–1544 (2003).

    Article  CAS  PubMed  Google Scholar 

  61. Wittenburg, N. et al. Presenilin is required for proper morphology and function of neurons in C. elegans. Nature 406, 306–309 (2000).

    Article  CAS  PubMed  Google Scholar 

  62. Geling, A., Steiner, H., Willem, M., Bally-Cuif, L. & Haass, C. A γ-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep. 3, 688–694 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Clark, I. E. et al. Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441, 1162–1166 (2006).

    Article  CAS  PubMed  Google Scholar 

  64. Cooley, L., Kelley, R. & Spradling, A. Insertional mutagenesis of the Drosophila genome with single P elements. Science 239, 1121–1128 (1988).

    Article  CAS  PubMed  Google Scholar 

  65. Gaiano, N. et al. Insertional mutagenesis and rapid cloning of essential genes in zebrafish. Nature 383, 829–832 (1996).

    Article  CAS  PubMed  Google Scholar 

  66. Bessereau, J. L. et al. Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line. Nature 413, 70–74 (2001).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  68. Morton, J., Davis, M. W., Jorgensen, E. M. & Carroll, D. Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells. Proc. Natl Acad. Sci. USA 103, 16370–16375 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Bedell, V. M. et al. In vivo genome editing using a high-efficiency TALEN system. Nature 491, 114–118 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Wood, A. J. et al. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Sander, J. D. et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat. Biotechnol. 29, 697–698 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Katsuyama, T. et al. An efficient strategy for TALEN-mediated genome engineering in Drosophila. Nucleic Acids Res. 41, e163 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. 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 

  76. 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 

  77. Liu, H. et al. TALEN-mediated gene mutagenesis in rhesus and cynomolgus monkeys. Cell Stem Cell 14, 323–328 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. 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). References 77 and 78 describe the successful generation of genetically modified non-human primates using genome-editing technologies in early embryos.

    Article  CAS  PubMed  Google Scholar 

  79. Chan, A. W., Chong, K. Y., Martinovich, C., Simerly, C. & Schatten, G. Transgenic monkeys produced by retroviral gene transfer into mature oocytes. Science 291, 309–312 (2001).

    Article  CAS  PubMed  Google Scholar 

  80. Yang, S. H. et al. Towards a transgenic model of Huntington's disease in a non-human primate. Nature 453, 921–924 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Sasaki, E. et al. Generation of transgenic non-human primates with germline transmission. Nature 459, 523–527 (2009).

    Article  CAS  PubMed  Google Scholar 

  82. Belmonte, J. C. et al. Brains, genes, and primates. Neuron 86, 617–631 (2015).

    Article  CAS  PubMed Central  Google Scholar 

  83. Xia, H., Mao, Q., Paulson, H. L. & Davidson, B. L. siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotechnol. 20, 1006–1010 (2002).

    Article  CAS  PubMed  Google Scholar 

  84. Kordasiewicz, H. B. et al. Sustained therapeutic reversal of Huntington's disease by transient repression of huntingtin synthesis. Neuron 74, 1031–1044 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Smith, R. A. et al. Antisense oligonucleotide therapy for neurodegenerative disease. J. Clin. Invest. 116, 2290–2296 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Garriga-Canut, M. et al. Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice. Proc. Natl Acad. Sci. USA 109, E3136–E3145 (2012).

    Article  PubMed  Google Scholar 

  87. Sweatt, J. D. The emerging field of neuroepigenetics. Neuron 80, 624–632 (2013).

    Article  CAS  PubMed  Google Scholar 

  88. Murlidharan, G., Samulski, R. J. & Asokan, A. Biology of adeno-associated viral vectors in the central nervous system. Front. Mol. Neurosci. 7, 76 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Burger, C., Nash, K. & Mandel, R. J. Recombinant adeno-associated viral vectors in the nervous system. Hum. Gene Ther. 16, 781–791 (2005).

    Article  CAS  PubMed  Google Scholar 

  90. Taymans, J. M. et al. Comparative analysis of adeno-associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain. Hum. Gene Ther. 18, 195–206 (2007).

    Article  CAS  PubMed  Google Scholar 

  91. Platt, R. J. et al. CRISPR–Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014). This paper describes how the CRISPR–Cas9 knock-in mouse can be used for cell type-specific gene editing in the brain.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

    Article  CAS  PubMed  Google Scholar 

  93. Akil, H. et al. Medicine. The future of psychiatric research: genomes and neural circuits. Science 327, 1580–1581 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Yin, L. et al. Multiplex conditional mutagenesis using transgenic expression of Cas9 and sgRNAs. Genetics (2015).

  95. Harris, J. A. et al. Anatomical characterization of Cre driver mice for neural circuit mapping and manipulation. Front. Neural Circuits 8, 76 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Dow, L. E. et al. Inducible in vivo genome editing with CRISPR–Cas9. Nat. Biotechnol. 33, 390–394 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  99. Pardo, B., Gomez-Gonzalez, B. & Aguilera, A. DNA repair in mammalian cells: DNA double-strand break repair: how to fix a broken relationship. Cell. Mol. Life Sci. 66, 1039–1056 (2009).

    Article  CAS  PubMed  Google Scholar 

  100. van Gent, D. C. & van der Burg, M. Non-homologous end-joining, a sticky affair. Oncogene 26, 7731–7740 (2007).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Peters, J. The role of genomic imprinting in biology and disease: an expanding view. Nat. Rev. Genet. 15, 517–530 (2014).

    Article  CAS  PubMed  Google Scholar 

  103. Kim, K. Y., Hysolli, E. & Park, I. H. Neuronal maturation defect in induced pluripotent stem cells from patients with Rett syndrome. Proc. Natl Acad. Sci. USA 108, 14169–14174 (2011).

    Article  PubMed  Google Scholar 

  104. Cheung, A. Y. et al. Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X-chromosome inactivation. Hum. Mol. Genet. 20, 2103–2115 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Marchetto, M. C. et al. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143, 527–539 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Harel, I. et al. A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate. Cell 160, 1013–1026 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Flowers, G. P., Timberlake, A. T., McLean, K. C., Monaghan, J. R. & Crews, C. M. Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease. Development 141, 2165–2171 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138–1142 (2015).

    Article  CAS  PubMed  Google Scholar 

  109. Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Qiu, P. et al. Mutation detection using Surveyor nuclease. Biotechniques 36, 702–707 (2004).

    Article  CAS  PubMed  Google Scholar 

  111. 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 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  116. Blasco, R. B. et al. Simple and rapid in vivo generation of chromosomal rearrangements using CRISPR/Cas9 technology. Cell Rep. 9, 1219–1227 (2014).

    Article  CAS  PubMed  Google Scholar 

  117. Maddalo, D. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516, 423–427 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. 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 

  119. Essletzbichler, P. et al. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res. 24, 2059–2065 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. 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). This paper describes the generation of mice that were genetically modified using Cas9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank R. Macrae for manuscript review and all members of the Zhang laboratory for discussions. M.H. is supported by the Human Frontiers Scientific Program. F.Z. is supported by the US National Institute of Mental Health (NIMH; grants 5DP1-MH100706 and 1R01-MH110049), the US National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; grant 5R01DK097768-03), the Poitras Center for Affective Disorders Research, the Vallee, Simons, Paul G. Allen Family, and New York Stem Cell Foundations, D. R. Cheng, T. Harriman and B. Metcalfe. F.Z. is a New York Stem Cell Foundation Robertson Investigator.

Author information

Authors and Affiliations

  1. Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, 02142, Massachusetts, USA

    Matthias Heidenreich & Feng Zhang

  2. McGovern Institute for Brain Research, Massachusetts Institute of Technology,

    Matthias Heidenreich & Feng Zhang

  3. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology,

    Matthias Heidenreich & Feng Zhang

  4. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Matthias Heidenreich & Feng Zhang

Authors
  1. Matthias Heidenreich
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Feng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Feng Zhang.

Ethics declarations

Competing interests

M.H. and F.Z. are named on patent applications related to work described herein. F.Z. is a founder of Editas Medicine and a scientific adviser for Editas Medicine and Horizon Discovery.

Related links

PowerPoint slides

Glossary

Functional genomics

The study of gene functions and interactions in relationship to RNA transcripts and protein products using genome-wide data, and often involving high-throughput methods.

RNA interference

(RNAi). A technique used to knock down the expression of a specific gene by introducing a double-stranded RNA molecule that complements the gene of interest and triggers the degradation of the target mRNA.

Homologous recombination

(HR). The exchange of homologous DNA strands between similar DNA molecules, an event that occurs naturally during meiosis to generate genetic variation. HR is used to direct error-free repair of DNA double-strand breaks induced by DNA nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) proteins.

Embryonic stem cells

(ESCs). Totipotent cells derived from embryos that can be genetically manipulated in vitro to generate transgenic, knock-in and knockout mice. ESCs can also be directed to differentiate into various cell types in vitro, including neurons and glial cells.

Induced pluripotent stem cells

(iPSCs). Pluripotent cells derived from reprogrammed differentiated adult cells; iPSCs have properties similar to those of embryonic stem cells and therefore can, in principle, be differentiated into all cell types of the body.

Epigenetic mechanisms

Multilayered cellular processes that modulate gene expression and function in response to interoceptive and environmental stimuli during development, adult life and ageing, including DNA methylation, post-translational histone modifications, ATP-dependent nucleosome and higher-order chromatin remodelling, non-coding RNA deployment and nuclear reorganization.

Liposomes

Lipid vesicles artificially formed by sonicating lipids in an aqueous solution. Liposomes can be packed with negatively charged molecules to deliver them into cells and are therefore promising vehicles for therapeutic applications.

Cre–loxP recombination

A site-specific recombination system derived from Escherichia coli bacteriophage P1. Two short DNA sequences (loxP sites) are engineered to flank the target DNA. Activation of the Cre recombinase enzyme catalyses recombination between the loxP sites, leading to excision of the intervening sequence.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Heidenreich, M., Zhang, F. Applications of CRISPR–Cas systems in neuroscience. Nat Rev Neurosci 17, 36–44 (2016). https://doi.org/10.1038/nrn.2015.2

Download citation

  • Published:

  • Issue Date:

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

This article is cited by

Search

Advanced 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