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Applications of CRISPR–Cas systems in neuroscience

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.

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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.

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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.

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Correspondence to Feng Zhang.

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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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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.

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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

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