One of the great potentials of zebrafish (Danio rerio) is to generate accurate models of human genetic diseases to recapitulate their clinical features, to understand the molecular mechanisms that underpin them, and to model treatments and disease management approaches1 as zebrafish and mammalian genes are highly conserved, and 70% of human disease genes have an ortholog in zebrafish2.
In addition, zebrafish are an attractive alternative to mouse models because they give rise to a large number of progeny and are amenable to high-throughput mutagenesis and drug screening approaches2. The process of gene targeting in zebrafish is not as laborious as it is in mice, and the maintenance costs are 10X cheaper per animal3. With the transformative CRISPR/Cas9 approach, it is now possible to target any number of genes in an efficient and high-throughput manner2.
There are many reviews discussing the use of CRISPR-based approaches and the various technological developments in zebrafish. We have compiled a list of most influential and informative articles based on our learnings to share with the zebrafish research community.
1. A beginner’s guide to understanding and implementing the genetic modification of zebrafish. Rafferty & Quinn, 2018.
A well rounded review provides a detailed roadmap outlining -numerous tools available for genetic modification in zebrafish, including the historical and experimental context in which they have been used. Methods including Tol2 transgenesis, TALENs and CRISPR/Cas9 are discussed. A particularly useful flowchart can help readers understand the numerous decision points and considerations involved in choosing the right approach for their particular experimental needs.
2. Precise Editing of the Zebrafish Genome Made Simple and Efficient. Hoshijima et al., 2016.
An excellent framework for the novice and expert genome engineer alike.The article outlines the design of a number of elegant genetic tools to facilitate CRISPR/Cas9 mediated knock-ins, as well as downstream screening and experimental uses. The focus here is on fluorescent tags, epitope tags, conditional tools, and transgenesis markers to help researchers screen for phenotypically silent edits.
3. Fishing for understanding: Unlocking the zebrafish gene editor’s toolbox. Simone et al., 2018.
If you’re looking for more mechanistic insight into gene editing in zebrafish, look no further than this review from Simone et al. The authors provide a detailed discussion of the different DNA repair pathways, template types, and experimental considerations that all factor into a successful gene editing endeavor. The various “designer nucleases” available for zebrafish gene editing applications, their advantages, and their limitations are also discussed.
4. Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9. Prykhozhij et al., 2018.
A thorough review of current methods around point mutation knock-ins in zebrafish using CRISPR/Cas9, including the detailed description of the screening workflow for identifying the rare precise editing events generated with current knock-in approaches.
5. Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish. Albadri et al., 2017.
An excellent overview of knock-ins in zebrafish. The article provides one of the most comprehensive side-by-side comparisons of donor template designs and knock-in strategies available for zebrafish at the time. A detailed discussion of the different repair pathways, template designs, and different reagent choices, as well as their limitations and paths forward for improving knock-in editing efficiencies in the future is very informative
6. CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments. Boel et al., 2018.
Boel et al. delve into the dizzying realm of ssODN-mediated knock-in repair with a comprehensive assessment of the impacts of different repair templates and design strategies on editing outcomes. Potential mechanisms of repair that lead to complex mutation patterns obtained with ssODN templates, as well as potential avenues for improvement on these methods are also discussed.
- Zebrafish knock-ins swim into the mainstream. Sergey V. Prykhozhij and Jason N. Berman.
- Expanding the CRISPR Toolbox in Zebrafish for Studying Development and Disease. Liu K, Petree C, Requena T, Varshney P, Varshney G.
- DNA-guided genome editing using structure-guided endonucleases. Varshney G, Burgess S.