Modeling Neuronal Diseases in Zebrafish in the Era of CRISPR

Abstract

Background: Danio rerio is a powerful experimental model for studies in genetics and development. Recently, CRISPR technology has been applied in this species to mimic various human diseases, including those affecting the nervous system. Zebrafish offer multiple experimental advantages: external embryogenesis, rapid development, transparent embryos, short life cycle, and basic neurobiological processes shared with humans. This animal model, together with the CRISPR system, emerging imaging technologies, and novel behavioral approaches, lay the basis for a prominent future in neuropathology and will undoubtedly accelerate our understanding of brain function and its disorders.

Objective: Gather relevant findings from studies that have used CRISPR technologies in zebrafish to explore basic neuronal function and model human diseases.

Methods: We systematically reviewed the most recent literature about CRISPR technology applications for understanding brain function and neurological disorders in D. rerio. We highlighted the key role of CRISPR in driving forward our understanding of particular topics in neuroscience.

Results: We show specific advances in neurobiology when the CRISPR system has been applied in zebrafish and describe how CRISPR is accelerating our understanding of brain organization.

Conclusion: Today, CRISPR is the preferred method to modify genomes of practically any living organism. Despite the rapid development of CRISPR technologies to generate disease models in zebrafish, more efforts are needed to efficiently combine different disciplines to find the etiology and treatments for many brain diseases.

Keywords: Brain disease models; CRISPR; Danio rerio; genome engineering; optogenetics; zebrafish..

Fig. (1)

Summary of the main strategies for zebrafish genome engineering using the CRISPR/Cas system. The typical time consumed in each procedure is indicated in red. A) sgRNA design using any of the available tools. In vitro synthesis of the sgRNAs and mRNA (or protein) from the Cas choice. Embryo microinjection at one-cell stage with a mix of single or multiple sgRNAs plus the following according to the purpose: for knockout, mRNA encoding Cas choice (or Cas protein); for knockin, mRNA encoding nuclease (or Cas protein) plus donor DNA; for gene regulation or cargo delivery, inactive dead Cas9 (dCas9) fused to an effector. Inside the cell, a complex is formed between the Cas protein, the sgRNA and the genomic DNA. B) The endonuclease generates a genomic DNA double-strand break, which is repaired by the endogenous DNA repair machinery by non-homologous end joining (NHEJ), causing insertions or deletions (InDels) that could disturb the open reading frame (knockout) or incorporate exogenous donor DNA into a homology-independent process at a chosen genomic locus (knockin). C) dCas9 can be used fused to an effector such as transcriptional repressors, activators, DNA methylases, or fluorescent proteins. In this case, the complex, instead of breaking the genomic DNA, allows gene regulation or target visualization. D) Phenotypes can be observed in the injected embryos after 1 to 5 days. Genotype screening can be performed in a portion of the mosaic F0 by a quick procedure such as TIDE (Tracking of Indels by Decomposition), HMA (heteroduplex mobility assay), or T7 endonuclease digestion, and then confirmed by PCR and sequencing. To keep the genotyped embryos alive, a ZEG device can be used instead of using the whole embryo for the analysis [67]. For a clear example of this procedure, see [101]. E) To avoid phenotype variability due to mosaicism, F1 can be analyzed in addition to the injected embryos (mosaics). F) To produce a mutant line, founder fish are outcrossed to wild type and then F1 inbred until homozygous are found. Genotyping is performed by fin-clip or skin swabbing [129]. If the homozygote is lethal, a heterozygous line can be maintained. For an example of this procedure, see [82].

Fig. (2)

Graphical representation of the genes related to neurobiology (in italics) edited by CRISPR/Cas to date and their associated phenotype in zebrafish. All genes are also listed in Table 1 with the corresponding reference. The main image in black illustrates the complete zebrafish (emphasizing the brain and spinal cord); genes related to the loss of midbrain-hindbrain boundaries, like pax2a or the optic tectum innervation (nox2); and genes related to neural tube formation like rfx4, spast, and prdm12b, among others, are included. Small zebrafish black head mainly represents the genes that produce microcephaly. A grey zebrafish shadow is a cyclops fish produced by nrd2/cyclops that includes glycine encephalopathy (gldc) and the morphological defects caused by disruption in tardbp1. Circles and squares with zebrafish larvae trajectories (in red) represent larvae motility with anesthesia (dbh) or the epileptic or autism phenotype.

Fig. (3)

CRISPR/Cas combined with optical tools in zebrafish. A) Generation of transgenic lines with CRISPR system expressing a fluorescent calcium indicator called genetically encoded calcium indicator (GECI) under promoter endogenous NET (norepinephrine transporter) to monitor noradrenergic system [119]. B) Optical control gene-editing protein system (TAEL). LOV (lightoxygen-voltage protein), HLH (helix-turn-helix DNA-binding domain), Ja (helix), TA photoactive transactivator (GAVPO Gal4 DNA binding domain and p65 activation domain), 5x-C120 (regulatory element termed) [122]. C) CRISPR/Cas combined with CRY2 light transcriptional activation. CIBI (cryptochrome interacting basic-helix-loop-helix 1), CRY2 (cryptochrome 2) [123].