Applications of CRISPR–Cas systems in neuroscience

Journal name:
Nature Reviews Neuroscience
Volume:
17,
Pages:
36–44
Year published:
DOI:
doi:10.1038/nrn.2015.2
Published online

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.

At a glance

Figures

  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.

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Affiliations

  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.

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  • 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.

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