Common Errors in Zebrafish Genetic Model Generation

Key Mistakes to Avoid for Successful Zebrafish Genetic Modeling

Creating zebrafish genetic models is a complex and intricate process that requires precision and attention to detail. However, many scientists encounter common errors that can hinder the successful generation of these models. In this article, we will explore some of these pitfalls and provide insights on how to avoid them based on scientific research and expertise.

Common errors in Zebrafish Genetic Model Generation

1. Improper selection of genetic targets and sgRNA design

One of the most critical steps in generating zebrafish knockout models is the design of sgRNAs. Improper design can lead to inefficient gene editing strategies leading to low targeting efficiency and/or off-target effects, resulting in wasted time and resources.

An appropriate design starts with a suitable selection of genetic targets. Failure to do so can compromise the entire genetic model generation process. 

Key considerations include:

• Choosing the wrong targets: genomic loci might have complex organization and many genes could be translated into different isoforms of the same protein. Placing the single guide RNAs (sgRNAs) in the correct site is the key to succeed in creating the desired mutation. 
• Choosing target sites that are not well-conserved can lead to ineffective genetic modifications. In some cases, there might be differences between the annotated genome and the sequence carried by the zebrafish strain in your fishroom. It is crucial to design and synthesize the optimal sgRNAs to correctly cut the desired targets.
• Overlooking Off-Target Effects: One critical pitfall is overlooking off-target effects in genome editing techniques like CRISPR/Cas9. Failure to assess and mitigate off-target mutations can lead to unintended genetic modifications that may impact the interpretation of research findings and the reliability of zebrafish genetic models.

SOLUTION: When designing a CRISPR/Cas9-based gene editing approach, ZeClinics’ experts perform a thorough analysis of the target locus to determine the optimal placement of the sgRNAs. In addition, they use bioinformatics tools to select well-conserved genetic target sites in silico, ensuring minimal predicted off-target effects. . Thus, we enhance the precision of gene editing processes.

2. Insufficient in vivo evaluation of somatic editing efficiency

For CRISPR/Cas9 knockout approaches, the in vivo cutting efficiency of the gene editing strategy must be assayed to determine the most efficient sgRNA. 

Frequent errors include:

• Poorly designed sgRNAs can result in low editing efficiency.
• Assuming that there is always a match between predicted and real efficiency: in some cases, the cutting efficiency of a guide might be lower than expected. 

SOLUTION: Our experts microinject an initial batch of embryos with different sgRNAs to be tested. Then, they use fully validated PCR primers and protocols for edit detection to confirm, in vivo, the somatic edit into the zebrafish genome and to select the best sgRNA to be used in the generation phase.

3. Inadequate plasmid design and validation

Generating a transgenic model is extremely efficient in zebrafish, however, this approach relies on a critical initial step: the design and generation of the correct transgenesis plasmid. Since the plasmid instructs the host animal's cells on how to express the new genetic material, ensuring  the accuracy of the sequence before attempting its insertion into the animal is crucial. 

Key consideration:

• The presence of sequence errors such as point mutations, misreads, or sequence deletions can lead to further complications downstream.
• Missing important parts of the plasmid such as the transposase recognition sites or the polyA signal might cause the failure of model generation.

SOLUTION: To implement strict quality control measures on the genetic starting material before involving the animals. The plasmid sequences we use are thoroughly verified before proceeding with the project. This verification process is called plasmid validation. By implementing these quality verification steps for the plasmids, we guarantee that our customers receive transgenic animals precisely designed to their specifications.

4. Suboptimal Injection Techniques

Microinjection is a delicate process that requires precision and expertise. Common issues include:

• Inconsistent Injection Volumes: Variability in injection volumes can lead to inconsistent expression levels of the genetic construct.
• Embryo Damage: Physical damage to embryos during injection can reduce survival rates and affect experimental outcomes.

SOLUTION: We standardize injection protocols and ensure proper training for personnel performing the injections. Our experts use consistent injection pressures and calibrated equipment to achieve uniform results.

Additional errors related to zebrafish genetic models generation

Beyond the generation of the genetic model itself, it's important to be aware of other possible experimental errors related to genetic models.

5. Issues with the experimental design

It is important to design the perfect experiment to validate each genetic model.  For instance, the use of proper controls are essential for validating experimental results. Common issues include the design of an experiment missing the correct negative controls: Without negative controls, it is difficult to ascertain whether observed effects are due to genetic modification or other factors.

SOLUTION: Design experiments with appropriate negative and positive controls. This includes unmodified wild-type zebrafish and known successful transgenic lines, respectively.

6. Inadequate Phenotypic Characterization

Phenotypic characterization is crucial for understanding the effects of genetic modifications. Common pitfalls include:

• Superficial Analysis: Relying solely on gross morphological changes without investigating underlying molecular or cellular mechanisms can lead to incomplete conclusions.
• Neglecting Behavioral Assessments: Behavioral changes can provide important insights into the effects of genetic modifications, but are often overlooked.

SOLUTION: Employ a comprehensive approach for the phenotypic characterization of your models, including molecular, cellular, and behavioral analyses. Use advanced imaging techniques and assays to obtain a complete picture of the phenotype.

Conclusion

The generation of zebrafish genetic models is a complex and competitive field. By recognizing and addressing common pitfalls, scientists can improve their chances of success. Proper design, execution, and validation of genetic constructs, coupled with rigorous phenotypic characterization, are key to advancing research in this area.

REFERENCES

[1] Hwang, W., Fu, Y., Reyon, D. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31, 227-229 (2013).

[2] Cornet, Carles et al. “Combining Zebrafish and CRISPR/Cas9: Toward a More Efficient Drug Discovery Pipeline.” Frontiers in pharmacology vol. 9 703. 3 Jul. 2018.

[3] Jao, L.E., Wente, S.R. & Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A. 110, 13904–13909 (2013).

[4] Ni, J., Wangensteen, K.J., Nelsen, D. et al. Active recombinant Tol2 transposase for gene transfer and gene discovery applications. Mobile DNA 7, 7 (2016).

[5] Howe, K., Clark, M.D., Torroja, C.F. et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 496, 498-503 (2013).

[6] Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio), 5th Edition. University of Oregon Press, 2007.

[7] Spence, R., Gerlach, G., Lawrence, C., & Smith, C. The behaviour and ecology of the zebrafish, Danio rerio. Biological Reviews. 83, 13-34 (2008).

[8] Kalueff, A.V., Stewart, A.M., & Gerlai, R. Zebrafish as an emerging model for studying complex brain disorders. Trends in Pharmacological Sciences. 35, 63-75 (2014).

[9] Dahm, R. & Geisler, R. Learning from small fry: the zebrafish as a genetic model organism for aquaculture fish species. Mar Biotechnol 8, 329–345 (2006).

[10] National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals. 8th edition. National Academies Press (US), 2011.

By Miriam Martínez

Miriam is a Human Biologist expert in neuropharmacology. After a master’s degree in Pharmaceutical and Biotech Industry, she obtained her PhD in Biomedicine from Pompeu Fabra University (Barcelona). During her doctorate, she focused her research on the behavioral analysis of animal models for neurophenotypical characterization. Since then, she has been working in the healthcare marketing and publicity sector, where she has contributed to developing marketing campaigns for several pharmaceutical brands. In 2021, she joined ZeClinics with a branding and marketing strategy focus.