Zebrafish Knockout Services

• Zebrafish Conditional Knockout Services
• Zebrafish Point Mutation Service

Complementary to mammalian models, the zebrafish system facilitates methods that are not possible (in vivo imaging of embryonic development), not practical (such as large-scale forward genetic screens), or not cost-effective (high-throughput chemical screens for drug discovery) in mice or rats. Zebrafish's exogenously fertilized embryos allow real-time, in vivo observation of development from the single-cell stage. Moreover, as a vertebrate, the zebrafish has many similarities with humans, including the nervous system, skin, blood and vasculature, cartilage and bone, liver, kidney, pancreas, gut, and innate and adaptive immune systems. This combination of features makes the zebrafish an exceptional model for studying development, human disease and for high-throughput drug studies.

Targeted genetic modifications stand as the single most desired methodology of the rapidly growing zebrafish market. The coming of CRISPR/Cas9-based genome modification has brought gene knockout and knock-in strategies to zebrafish researchers. The CRISPR/Cas9 system represents an important step forward towards achieving precise and targeted gene disruption. Being readily applicable for the generation of knockout loci in a great variety of animal models, this technology has resulted in significant advances in the fields of drug discovery. Targeted gene editing with CRISPR/Cas9 system has revolutionized reverse genetic manipulation of zebrafish and other model organisms.

Zebrafish Targeted Knockout

Generating knockout alleles in zebrafish by CRISPR/Cas9 is rapid and not difficult. Zebrafish lines carrying homozygous CRISPR/Cas9 mutant alleles can be obtained in only two generations or less. The selection of the sgRNA target sequence is guided by heuristic rules developed from analysis of the cutting efficiencies of different sgRNA molecules in vivo. The sgRNAs are injected directly into the zebrafish zygote either with in vitro-synthesized mRNA encoding a nuclear localized Cas9. Low fidelity DSB repair occurs at each target of each diploid cell independently leading to the generation of distinct alleles.

Figure 1. Strategy for Zebrafish Genome Engineering with CRISPR/Cas9. (Li M, et al. 2016)

Using CRISPR/Cas9, Creative Biogene can delete integral domains or the entire coding sequence of a gene in zebrafish, depending on gene size. We have generated lesions ranging from small indels to full gene deletions. Our customized zebrafish knockout models include:

• Transient Knockout: This knockout approach allows the phenotypic screening of genes and pathways, providing a fast method for performing target validation for disease-relevant genes identified by genomic strategies.
• Isogenic Stable Knockout: Many zebrafish mutant models have been developed through CRISPR/Cas9. These zebrafish disease models can be applied to analyze the pathogenic effect of a given mutation or test a battery of candidate drugs before proceeding to further preclinical trials with mammalian animal models.
• Tissue-Specific Knockout: This method offers the possibility to study gene function in specific tissues. Besides, these models allow simultaneous gene inactivation and mutant cell fate analysis through fluorescent cell tracing.

Creative Biogene provides customized zebrafish knockout model building services, including expert research design, in vitro sgRNA design synthesis, CRISPR vector construction and final zebrafish, allowing you to precisely control the expression of target genes in zebrafish. Our zebrafish knockout services will help advance your developmental and reproductive research, disease research, preclinical drug discovery and toxicology programs.

References

1. Cornet C, et al. Combining zebrafish and CRISPR/Cas9: toward a more efficient drug discovery pipeline. Frontiers in pharmacology, 2018, 9: 703.
2. Bedell V M, Ekker S C. Using engineered endonucleases to create knockout and knockin zebrafish models. Chromosomal Mutagenesis. Humana Press, New York, NY, 2015: 291-305.
3. Li M, et al. Zebrafish genome engineering using the CRISPR–Cas9 system. Trends in Genetics, 2016, 32(12): 815-827.
4. Albadri S, et al. CRISPR/Cas9-mediated knockin and knockout in zebrafish. Genome Editing in Neurosciences. Springer, Cham, 2017: 41-49.
5. Chang N, et al. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell research, 2013, 23(4): 465-472.

For research use only. Not intended for any clinical use.