October 1, 2016 (Vol. 36, No. 17)

Using Zebrafish as an Ideal Model Organism for Functional Gene Knockout Studies

Gene editing is a powerful molecular biology tool that has been rapidly integrated into life sciences research, offering great potential to transform our understanding of disease and future therapies. One such gene-editing system, CRISPR-Cas9, allows researchers to quickly edit genes for functional gene knockouts in mammalian, fish, and plant genomes.

Based on a prokaryotic immune system-like mechanism, the CRISPR-Cas9 system uses a combination of the Cas9 protein, CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to edit the genome, allowing researchers to study disease pathways in a variety of model organisms.

The CRISPR-Cas9 system requires exogenous Cas9 nuclease to be delivered into the cell with a guide RNA; this can be accomplished through transfection of a Cas9 gene expression plasmid, mRNA or protein, or through transduction with active lentiviral particles. DNA-based Cas9 constructs are used in many applications but they can result in unwanted integration events. Successful lentiviral delivery results in integration of the Cas9 expression cassette into the cell’s genome, and transient transfection of a Cas9 plasmid may also result in permanent and random insertion of the vector sequence into the genome.

An alternative approach that uses Cas9 mRNA or protein avoids unwanted integration and, in combination with synthetic crRNA and tracrRNA, results in a completely DNA-free gene-editing system. It is transient, has fewer off-target effects, and can be less toxic. An additional benefit of using GE Healthcare Dharmacon™ Edit-R™ synthetic crRNAs is the design algorithm by which they are generated, which accurately identifies and avoids sequences that might result in off-target editing, while maintaining high functionality, using data derived from functional gene knockout laboratory experiments.

The zebrafish (Danio rerio) is quickly becoming a preferred organism in biomedical research as a result of its high degree of sequence and functional homology with humans, its low cost, and the relative ease of use in the laboratory. An additional feature that makes the zebrafish model ideal for use in genetics studies is its natural rapid external development, which makes it suitable for studying early developmental events.

Zebrafish research has expanded to a wide variety of basic science and clinical research settings, including modeling human genetic disease.1 Zebrafish as a model for human disease is enhanced by the considerable genomic resources that exist, including a database of characterized mutant lines,2 and a complete and annotated genome.

This study demonstrates successful gene editing using DNA-free CRISPR-Cas9 reagents for gene knockout in zebrafish targeting green fluorescent protein (GFP), a jellyfish (Aequorea victoria)-derived protein whose expression is simple to detect by fluorescent microscopy techniques.

The Study

To determine the efficacy of a DNA-free CRISPR-Cas9 genome engineering platform in zebrafish, embryos expressing both GFP and red fluorescent protein (RFP) were either microinjected with Edit-R Cas9 Nuclease mRNA and synthetic crRNA:tracrRNA, using a custom crRNA targeting GFP3 or injected with Cas9 mRNA only (control group). Survival was assessed one and two days after fertilization, and in both groups the injected embryos had a high survival rate.

Two days post-fertilization, genomic DNA was extracted from eight of the embryos and analyzed for gene-editing efficiency using a mismatch detection assay, which detects mismatches due to insertions and deletions (indels) in DNA where CRISPR gene editing had been carried out. When all three components of the CRISPR-Cas9 system were injected, targeted DNA cleavage was achieved in 75% of the zebrafish embryos analyzed, and the gene-editing efficiency was 9-20%.

Importantly, the analyzed embryos that showed gene editing likely have mutations of the germ cell and can be used for generation of a stable transgenic line for further scientific interrogation.4

Detecting a functional gene knockout of GFP was performed by imaging zebrafish microinjected with the DNA-free CRISPR system targeting this gene (Cas9 mRNA and synthetic crRNA:tracrRNA), and comparing them to control embryos, with RFP as a control gene, which would not be influenced by the CRISPR-Cas9 gene editing. Control embryos continued to express both GFP and RFP using confocal imaging (Figure 1A), whereas a loss or decrease in GFP fluorescence was observed in CRISPR-Cas9-targeted zebrafish embryos (Figure 1B). In the latter group RFP continued to be expressed normally, confirming a successful and specific gene knockout.

Zebrafish are an excellent model system for genome-engineering studies, and can be used in conjunction with a completely DNA-free gene-editing system and in vivo microinjection of zebrafish embryos to knock out a gene, in this study GFP.

Gene editing with DNA-free CRISPR-Cas9 components reduces potential off-targets, and illustrates the possibility of using CRISPR-Cas9 gene editing in model systems to find correlations with human diseases.

Figure 1. GFP knockout in vivo using Edit-R Cas9 mRNA & synthetic crRNA:tracrRNA. Fluorescence microscopy shows dorsal view of zebrafish neural tube 24 h post-fertilization. (A) Neural crest cells expressing both GFP and RFP in zebrafish single-cell embryos after injection with only Cas9 mRNA (control group). (B) Neural crest cells in embryos injected with Cas9 and crRNA:tracrRNA targeting transgenic GFP (experimental group) display mosaic GFP expression as a result of functional protein knockout in some cells. (Scale bars = 37 µM).

1. G.J. Lieschke and P.D. Currie. Animal models of human disease: zebrafish swim into view. Nat. Rev. Genet. 8, 353–367 (2007).
2. The Zebrafish Model Organism Database, zfin.org.
3. L.-E. Jao, S. R. Wente and W. Chen. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl. Acad. Sci. U.S.A. 110, 13904-13909 (2013).
4. Microinjection of zebrafish embryos using Dharmacon™ Edit-R™ Cas9 Nuclease mRNA and synthetic crRNA and tracrRNA for gene engineering. A.J. Blasky*, A. Haas, J.A. Schiel, R. Prekeris*, M.L. Kelley. * University of Colorado, Denver, CO. Dharmacon, Lafayette, CO.

Amanda Haas ([email protected]) is a scientist at GE Healthcare’s Life Sciences business. GE, imagination at work, and GE monogram are trademarks of General Electric Company. Dharmacon and Edit-R are trademarks of GE Healthcare. All other trademarks are the property of General Electric Company or one of its subsidiaries. 

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