Applications of CRISPR-Cas systems in neuroscience

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.

Figure 1. Genome editing applications of CRISPR-Cas9

(a) Non-homologous end-joining (NHEJ) and homology-directed repair (HDR) after DNA double-strand break (red arrowheads) induced by zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) (left) and Cas9 (right). ZFNs and TALENs recognize their DNA binding site via protein domains (indicated in blue) that can be modularly assembled for each DNA target sequence. Cas9 recognizes its DNA binding site via RNA-DNA interactions mediated by the short guide RNA (sgRNA), which can be easily designed and cloned. The error-prone NHEJ repair pathway can result in introduction of indel mutations that can lead to a frame shift, introduction of a premature stop codon and consequently gene knockout. The alternative HDR repair pathway^{, –} 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 rearrangements^, . (c) Two proximate sgRNAs guide Cas9 to induce DNA cleavage at two different loci of the same gene, introducing large deletions^, . (d) The nuclease inactivated version of Cas9 (dead Cas9 (dCas9)) can be fused to different functional enzymatic domains in order to mediate transcriptional control, epigenetic modulation editing, or fluorescent DNA labeling of specific genetic loci^–.

Figure 2. Methods for generating genetically modified rodents

Comparison of the timelines of traditional gene targeting using classic homologous recombination (HR) in embryonic stem cells (ESCs) or Cas9 in one-cell embryos. (a) There are two main time- and cost-intensive phases of the HR approach. The first, is the design and cloning the targeting vector, ESC transduction and selection, and generation of chimeras. The second is the backcrossing of mice to a desired background and/or crossbreeding in order to generate multiple genetically modified animals. (b) By contrast, cloning of sgRNA into targeting vector, verification of sgRNA on-target efficiency, Cas9/sgRNA microinjection, and founder identification are relatively easy and fast^, . Because embryos can be obtained from any mouse strain and multiple genes can be targeted simultaneously, no genetic backcrossing and crossbreeding is required.

Figure 3. In vivo genome editing strategies using viral delivery of Cas9 in the mammalian brain

Cas9 nucleases enable precise in vivo genome editing of specific cell types in the mammalian brain on a relatively short time-scale. 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 (AAV). sgRNA can then be stereotactically delivered into the brain of mice that express endogenous Cas9 expression (Cas9 mice, (left)), or together with Cas9 into wildtype mice or rats, aged and disease models, or reporter lines. In vivo genome editing in the brain is not limited to rodents and can be theoretically applied to other mammalian systems including non-human primates (right). hSyn: human Synapsin promoter; GFAP: glial fibrillary acidic protein.

Figure 4. In vitro application of Cas-based genome editing in human induced pluripotent stem cells (iPSCs)

(a) Evaluation of disease candidate genes from large-population genome-wide association studies (GWAS). Human primary cells such as neurons are not easily available and are difficult to expand in culture. By contrast, iPSCs derived from somatic cells (such as fibroblasts) of healthy individuals or patients with neurological disorders can be differentiated into neurons and cultured in vitro^–. Disease candidate genes can be examined in two ways. Site-specific homologous recombination (HDR) of the candidate gene using Cas nucleases can be applied in disease-affected cells (top). 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 (bottom). 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’s or Parkinson’s disease can be systematically evaluated using Cas-mediated single and multiplex genome editing. This may enable the 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.