Zebrafish Knockin Services

• Tol2 Transposon-based System Service
• Zebrafish Reporter Lines Generation

The zebrafish is frequently used for both basic developmental biology research and human disease modeling due to its transparency, fecundity, and the availability of well-developed genetic and cell biological tools. Recently, with the development of genome editing technologies, the possible integration of exogenous genes into any given loci and the analysis of their function in the living animal have dramatically improved. Before this recent explosion in the field of knockin generation, conventional transgenic zebrafish lines were generated by Tol2-mediated transgenesistechnology, which has successfully allowed the making of hundreds of new reporter lines essential to the study of particular gene functions in vivo.

With its variety of applications, the CRISPR/Cas9 genome editing technology has been rapidly developing in recent years. Strategies employing CRISPR/Cas9 are now used in various species with one of the greatest advantages being the ability to introduce specific genomic modifications. In the zebrafish community, knockout reports are constantly increasing but insertion studies have been so far more challenging. As a leading provider of zebrafish service, we have developed a CRISPR/Cas9-mediated efficient knockin strategy for zebrafish, which can be widely applied for tagging endogenous proteins and labeling different cell types.

Zebrafish Knockin

Our expert's team of zebrafish research provides expertise in transgenic manipulation and zebrafish biology for the generation of zebrafish knock-in lines, and subsequent phenotypic analysis for custom drug screening.

• HDR-Mediated Knockin: Homology directed repair allows the precise integration of DNA fragments. Small modifications such as LoxP integration or single nucleotide editing can be achieved by providing a single-stranded oligodeoxynucleotide (ssODN) as donor DNA for CRISPR/Cas9-mediated HDR.
• NHEJ-Mediated Knockin: Targeted insertion of exogenous DNA fragments can be achieved by making use of NHEJ repair after DSB events. This approach could allow the generation of more precise reporter lines for several genes or signaling pathways. For instance, that could allow to identify drugs altering Notch, BMP, or WNT signaling, which is important players in development, but also in cancer progression.
• DNA Base Editing: A strategy for precise single “base editing” (BE) has been implemented in zebrafish. BE system is based on the fusion of a cytidine deaminase to a Cas9 nickase (nCas9), which permits a DSB-independent irreversible conversion of one targeted base to another. The use of base editors will allow the development of several zebrafish disease models mimicking specific human polymorphisms.

Creative Biogene provides customized zebrafish knockin model services, including sgRNA design, vector construction, final knock in zebrafish generation, screening and identification service. Combining the advantages of zebrafish and the CRISPR/Cas9 system, we will provide a valuable tool for streamlining the generation of zebrafish models mimicking human disease, the validation of novel drug targets and the discovery of new therapeutics.

References

1. Kawahara A. CRISPR/Cas9-Mediated Targeted Knockin of Exogenous Reporter Genes in Zebrafish. Genome Editing in Animals. Humana Press, New York, NY, 2017: 165-173.
2. Albadri S, et al. CRISPR/Cas9-mediated knockin and knockout in zebrafish. Genome Editing in Neurosciences. Springer, Cham, 2017: 41-49.
3. Cornet C, et al. Combining zebrafish and CRISPR/Cas9: toward a more efficient drug discovery pipeline. Frontiers in pharmacology, 2018, 9: 703.
4. Prykhozhij S V, et al. Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9. Nucleic acids research, 2018, 46(17): e102-e102.

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