Applications of CRISPR–Cas systems in neuroscience

Journal name:
Nature Reviews Neuroscience
Year published:
Published online


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.

At a glance


  1. Genome-editing applications of CRISPR-Cas9.
    Figure 1: Genome-editing applications of CRISPR–Cas9.

    a | Non-homologous end-joining (NHEJ) and homology-directed repair (HDR) after a DNA double-strand break (DSB) is induced by zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9). ZFNs and TALENs recognize their DNA-binding site via protein domains that can be modularly assembled for each DNA target sequence. Cas9 recognizes its DNA-binding site via RNA–DNA interactions mediated by the short single-guide RNA (sgRNA), which can be easily designed and cloned. The error-prone NHEJ repair pathway53 can result in the introduction of insertion or deletion (indel) mutations that can lead to a frame shift, the introduction of a premature stop codon and, consequently, gene knockout. The alternative repair pathway, HDR14, 47, 48, 49, 50, 51, 52, 53, can be used to introduce precise genetic modifications if a homologous DNA template is present. b | Two different sgRNAs guide Cas9 to induce DNA cleavage at two different genes, resulting in chromosomal rearrangements116, 117. c | Two proximate sgRNAs guide Cas9 to induce DNA cleavage at two different loci of the same gene, introducing large deletions118, 119. d | The nuclease-inactivated version of Cas9 (dead Cas9 (dCas9)) can be fused to different functional enzymatic domains to mediate transcriptional control, epigenetic modulation or fluorescent DNA labelling of specific genetic loci30, 31, 32, 33, 34, 35, 36. HR, homologous recombination; M, methyl group.

  2. Using Cas9 to generate genetically modified rodents and for in vivo genome editing.
    Figure 2: Using Cas9 to generate genetically modified rodents and for in vivo genome editing.

    a,b | Comparison of the timelines of traditional gene targeting using classic homologous recombination (HR) in embryonic stem cells (ESCs; part a) and clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) gene targeting in one-cell embryos (part b). There are two main time- and cost-intensive phases of the HR approach. First, the design and cloning of the targeting vector, ESC transduction and selection, and generation of chimaeras. Second, the backcrossing of mice to a desired background and/or cross-breeding to generate multiple genetically modified animals. By contrast, cloning of short single-guide RNA (sgRNA) into a targeting vector, verification of sgRNA on-target efficiency (through the surveyor nuclease assay or sequencing), Cas9–sgRNA microinjection and founder identification are relatively easy and fast120. Because embryos can be obtained from any mouse strain and multiple genes can be targeted simultaneously, genetic backcrossing and cross-breeding are not required. c | Cas9 nucleases also enable precise in vivo genome editing of specific cell types in the mammalian brain on a relatively short timescale. Cas9 is cloned under the control of cell type-specific promoters, and sgRNA efficiency is validated in vitro before being packaged into viral vectors, such as adeno-associated viruses (AAVs). sgRNA can then be stereotactically delivered into the brains of mice that have endogenous Cas9 expression (Cas9 mice)91, or the sgRNA can be delivered together with Cas9 into wild-type mice41 or rats, aged animals, disease models or reporter lines. In vivo genome editing in the brain is not limited to rodents and can theoretically be applied to other mammalian systems, including non-human primates. GFAP, glial fibrillary acidic protein; Neo, neomycin anitibiotic selection marker; SYN, human synapsin promoter.

  3. In vitro applications of Cas9 in human iPSCs.
    Figure 3: In vitro applications of Cas9 in human iPSCs.

    a | Evaluation of disease candidate genes from large-population genome-wide association studies (GWASs). Human primary cells, such as neurons, are not easily available and are difficult to expand in culture. By contrast, induced pluripotent stem cells (iPSCs) derived from somatic cells (such as fibroblasts) of healthy individuals or patients with neurological disorders can be differentiated into neurons and cultured in vitro8, 9, 10, 11, 12. Disease candidate genes can be examined in two ways. Site-specific homologous recombination (HR) of the candidate gene using clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) nucleases can be applied in disease-affected cells (left). If this rescues disease phenotypes (as for candidate gene B in the example shown), the validity of the candidate gene is confirmed. Alternatively, candidate genes can be mutated in healthy cells (right). Where this recapitulates disease pathogenesis in vitro (as in the case of candidate gene B), the validity of the candidate gene is confirmed. b | The contribution of specific genetic loci to multigenic disorders, such as Alzheimer or Parkinson diseases, can also be systematically evaluated using Cas-mediated single and multiplex genome editing. This may enable dissection of possible synergistic effects (as shown for candidate genes A and B) and screening for functional correlations between disease phenotypes and distinct gene mutations. sgRNA, single-guide RNA.


  1. Lewis, E. B. & Bacher, F. Methods for feeding ethyl methane sulfonate (EMS) to Drosophila males. Drosoph. Inf. Serv. 43, 193194 (1968).
  2. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806811 (1998).
  3. St Johnston, D. The art and design of genetic screens: Drosophila melanogaster. Nat. Rev. Genet. 3, 176188 (2002).
  4. Jorgensen, E. M. & Mango, S. E. The art and design of genetic screens: Caenorhabditis elegans. Nat. Rev. Genet. 3, 356369 (2002).
  5. Patton, E. E. & Zon, L. I. The art and design of genetic screens: zebrafish. Nat. Rev. Genet. 2, 956966 (2001).
  6. Thomas, K. R. & Capecchi, M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503512 (1987).
  7. Oddo, S. et al. Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Aβ and synaptic dysfunction. Neuron 39, 409421 (2003).
  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, 20542062 (2012).
  9. Yagi, T. et al. Modeling familial Alzheimer's disease with induced pluripotent stem cells. Hum. Mol. Genet. 20, 45304539 (2011).
  10. Ryan, S. D. et al. Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription. Cell 155, 13511364 (2013).
  11. Soldner, F. et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146, 318331 (2011).
    References 10 and 11 combine ZFN-mediated genome-editing and human-stem-cell technologies for studying neurological disorders in vitro.
  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, 316328 (2015).
  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, 11561160 (1996).
  14. Bibikova, M. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell. Biol. 21, 289297 (2001).
    This early study shows the use of ZFNs in Xenopus laevis for stimulating HR.
  15. Urnov, F. D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646651 (2005).
  16. Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 15091512 (2009).
  17. Moscou, M. J. & Bogdanove, A. J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).
  18. Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757761 (2010).
  19. Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143148 (2011).
    This paper uses an improved TALEN architecture to introduce gene knockouts in human cells.
  20. Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S. D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 25512561 (2005).
  21. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 17091712 (2007).
    This work provides the first experimental demonstration of the adaptive immune function of the CRISPR–Cas9 system in bacteria.
  22. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602607 (2011).
  23. Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 6771 (2010).
    This paper demonstrates that Cas9 facilitates RNA-guided DNA cleavage in bacteria.
  24. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819823 (2013).
  25. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823826 (2013).
    References 24 and 25 describe the successful harnessing of the CRISPR–Cas9 system for editing the mammalian genome in cell lines.
  26. Makarova, K. S. & Koonin, E. V. Annotation and classification of CRISPR–Cas systems. Methods Mol. Biol. 1311, 4775 (2015).
  27. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759771 (2015).
  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, 1462814633 (1998).
  29. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149153 (2011).
  30. Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442451 (2013).
  31. Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 11731183 (2013).
  32. Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472476 (2013).
  33. Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401403 (2015).
  34. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583588 (2015).
  35. Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510517 (2015).
  36. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 14791491 (2013).
  37. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816821 (2012).
  38. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827832 (2013).
  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).
  40. Incontro, S., Asensio, C. S., Edwards, R. H. & Nicoll, R. A. Efficient, complete deletion of synaptic proteins using CRISPR. Neuron 83, 10511057 (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.
  41. Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR–Cas9. Nat. Biotechnol. 33, 102106 (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.
  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, 625636 (2014).
    This paper describes a conditional-knockout strategy using Cas9 in Caenorhabditis elegans for studying gene function in neural development.
  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, 535540 (2015).
    This paper represents a useful application of Cas9 for studying neurodevelopmental processes on a large scale in zebrafish.
  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, 142153 (2014).
  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, 1390413909 (2013).
  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.
  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, 451460 (1992).
  48. Rudin, N., Sugarman, E. & Haber, J. E. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics 122, 519534 (1989).
  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, 480484 (1992).
  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, 12921303 (1992).
  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, 51725177 (1998).
  52. Johnson, R. D., Liu, N. & Jasin, M. Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination. Nature 401, 397399 (1999).
  53. Bibikova, M., Beumer, K., Trautman, J. K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003).
  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, 80968106 (1994).
  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, 60646068 (1994).
  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, E2579E2586 (2012).
    This paper, along with reference 37, characterizes Cas9-mediated DNA cleavage in vitro.
  57. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186191 (2015).
  58. Brenowitz, E. A. & Zakon, H. H. Emerging from the bottleneck: benefits of the comparative approach to modern neuroscience. Trends Neurosci. 38, 273278 (2015).
  59. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494498 (2001).
  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, 15391544 (2003).
  61. Wittenburg, N. et al. Presenilin is required for proper morphology and function of neurons in C. elegans. Nature 406, 306309 (2000).
  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, 688694 (2002).
  63. Clark, I. E. et al. Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441, 11621166 (2006).
  64. Cooley, L., Kelley, R. & Spradling, A. Insertional mutagenesis of the Drosophila genome with single P elements. Science 239, 11211128 (1988).
  65. Gaiano, N. et al. Insertional mutagenesis and rapid cloning of essential genes in zebrafish. Nature 383, 829832 (1996).
  66. Bessereau, J. L. et al. Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line. Nature 413, 7074 (2001).
  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, 1982119826 (2008).
  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, 1637016375 (2006).
  69. Doyon, Y. et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat. Biotechnol. 26, 702708 (2008).
  70. Bedell, V. M. et al. In vivo genome editing using a high-efficiency TALEN system. Nature 491, 114118 (2012).
  71. Wood, A. J. et al. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307 (2011).
  72. Sander, J. D. et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat. Biotechnol. 29, 697698 (2011).
  73. Katsuyama, T. et al. An efficient strategy for TALEN-mediated genome engineering in Drosophila. Nucleic Acids Res. 41, e163 (2013).
  74. Sander, J. D. & Joung, J. K. CRISPR–Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347355 (2014).
  75. Carbery, I. D. et al. Targeted genome modification in mice using zinc-finger nucleases. Genetics 186, 451459 (2010).
  76. Geurts, A. M. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433 (2009).
  77. Liu, H. et al. TALEN-mediated gene mutagenesis in rhesus and cynomolgus monkeys. Cell Stem Cell 14, 323328 (2014).
  78. Niu, Y. et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156, 836843 (2014).
    References 77 and 78 describe the successful generation of genetically modified non-human primates using genome-editing technologies in early embryos.
  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, 309312 (2001).
  80. Yang, S. H. et al. Towards a transgenic model of Huntington's disease in a non-human primate. Nature 453, 921924 (2008).
  81. Sasaki, E. et al. Generation of transgenic non-human primates with germline transmission. Nature 459, 523527 (2009).
  82. Belmonte, J. C. et al. Brains, genes, and primates. Neuron 86, 617631 (2015).
  83. Xia, H., Mao, Q., Paulson, H. L. & Davidson, B. L. siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotechnol. 20, 10061010 (2002).
  84. Kordasiewicz, H. B. et al. Sustained therapeutic reversal of Huntington's disease by transient repression of huntingtin synthesis. Neuron 74, 10311044 (2012).
  85. Smith, R. A. et al. Antisense oligonucleotide therapy for neurodegenerative disease. J. Clin. Invest. 116, 22902296 (2006).
  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, E3136E3145 (2012).
  87. Sweatt, J. D. The emerging field of neuroepigenetics. Neuron 80, 624632 (2013).
  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).
  89. Burger, C., Nash, K. & Mandel, R. J. Recombinant adeno-associated viral vectors in the nervous system. Hum. Gene Ther. 16, 781791 (2005).
  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, 195206 (2007).
  91. Platt, R. J. et al. CRISPR–Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440455 (2014).
    This paper describes how the CRISPR–Cas9 knock-in mouse can be used for cell type-specific gene editing in the brain.
  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, 7380 (2015).
  93. Akil, H. et al. Medicine. The future of psychiatric research: genomes and neural circuits. Science 327, 15801581 (2010).
  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).
  96. Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR–Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198200 (2015).
  97. Dow, L. E. et al. Inducible in vivo genome editing with CRISPR–Cas9. Nat. Biotechnol. 33, 390394 (2015).
  98. Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139142 (2015).
  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, 10391056 (2009).
  100. van Gent, D. C. & van der Burg, M. Non-homologous end-joining, a sticky affair. Oncogene 26, 77317740 (2007).
  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, 539546 (2013).
  102. Peters, J. The role of genomic imprinting in biology and disease: an expanding view. Nat. Rev. Genet. 15, 517530 (2014).
  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, 1416914174 (2011).
  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, 21032115 (2011).
  105. Marchetto, M. C. et al. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143, 527539 (2010).
  106. Harel, I. et al. A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate. Cell 160, 10131026 (2015).
  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, 21652171 (2014).
  108. Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 11381142 (2015).
  109. Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121131 (2015).
  110. Qiu, P. et al. Mutation detection using Surveyor nuclease. Biotechniques 36, 702707 (2004).
  111. Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 13801389 (2013).
  112. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833838 (2013).
  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, 279284 (2014).
  114. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187197 (2015).
  115. Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR–Cas9 off-target effects in human cells. Nat. Methods 12, 237243 (2015).
  116. Blasco, R. B. et al. Simple and rapid in vivo generation of chromosomal rearrangements using CRISPR/Cas9 technology. Cell Rep. 9, 12191227 (2014).
  117. Maddalo, D. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516, 423427 (2014).
  118. Xiao, A. et al. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res. 41, e141 (2013).
  119. Essletzbichler, P. et al. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res. 24, 20592065 (2014).
  120. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910918 (2013).
    This paper describes the generation of mice that were genetically modified using Cas9.

Download references

Author information


  1. Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts 02142, 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, Massachusetts 02139, USA.

    • Matthias Heidenreich &
    • Feng Zhang

Competing interests statement

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.

Corresponding author

Correspondence to:

Author details

  • Matthias Heidenreich

    Matthias Heidenreich received his Ph.D. from the Freie Universität Berlin, Germany, carrying out his doctoral and early postdoctoral research at the Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin. He is currently a postdoctoral research fellow in the laboratory of Feng Zhang at the Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, USA, and the McGovern Institute for Brain Research at MIT. His research is focused on the development of new genome-engineering technologies for studying higher cognitive function and neurological disorders.

  • Feng Zhang

    Feng Zhang is an investigator at the McGovern Institute for Brain Research at Massachusetts Institute of Technology (MIT), Cambridge, USA, a core member of the Broad Institute of MIT and Harvard, Cambridge, USA, the W. M. Keck Career Development Professor in Biomedical Engineering and an assistant professor in the MIT Departments of Brain and Cognitive Sciences and Biological Engineering. He played an integral part in the development of optogenetics and CRISPR–Cas (clustered regularly interspaced short palindromic repeat–CRISPR-associated protein) genome-engineering technologies. His work is focused on understanding the mechanisms of neurological and psychiatric disorders through the development and application of novel molecular technologies. Feng Zhang's homepage.

Additional data