Can you Play with the Zebrafish Genome for Drug Discovery?

ZeClinics can do it by using different genome editing approaches.

By Flavia De Santis, Head of Genetics Department at ZeClinics.

Flavia obtained her master's degree in medical biotechnologies from the University of Bari (Italy), then she pursued her scientific education at the Institut Curie (Paris), where she obtained a Ph.D. in neuronews. During her doctorate, she focused her research on vertebrate neural circuit development and, in parallel,  worked on the implementation of innovative CRISPR/Cas9-based genome engineering approaches. Since she joined ZeClinics as a senior scientist, she has been coordinating a wide range of genetics services while working in the development of cutting-edge technologies to achieve a more efficient generation of zebrafish models for #therapeutic target validation.

In the previous article of this news, we explained why zebrafish is a powerful and valuable model for biomedical research. In this post, we will deepen this description, explaining how we can play with its genome to answer specific scientific questions. Indeed, genetically modified zebrafish can be employed in different fields of basic and applied research. Its applications include (but are not limited to) the validation of new druggable targets, the identification of the genetic cause of rare diseases, the dissection of molecular pathways involved in developmental or degenerative processes, the understanding of the regulation of key regenerative mechanisms.

In the past years, researchers established different experimental strategies to investigate the genetic causes of human diseases. Among these, there is the generation of genetic models in which selected genes are disrupted and inactivated. The rationale behind this method is quite simple: to be properly realized, every process requires a specific set of gears; if one essential item is removed, the entire process will be affected and could not be completed successfully. Biological processes (like a heart beating, food digestion, or neuronal communication) are not different: as they rely on a well-defined combination of genes, the inactivation of an essential gene will cause deficiencies and defects that will impair their execution (in the case of human disease, these defects are no more than the symptoms of the disease).

Independently from the biological problem, they want to solve, to develop a good genetic model, scientists need:

  • A system to introduce mutations in the target gene (or genes)
  • A way to observe the biological process of interest (to be able to spot the potential defects deriving from gene inactivation).

In zebrafish, these two aspects can be perfectly combined, making this tiny fish an ideal genetic model. In the next paragraphs, we will explain how ZeClincs takes advantage of these unique features to create genetic models. In the past decade, the field of genome engineering was revolutionized by the introduction of the CRISPR/Cas9 technology, a real breakthrough that earned her pioneers (J. Doudna and E. Charpentier) the Nobel prize for chemistry. Zebrafish genome can be targeted by the CRISPR/Cas9 machinery, making it possible to inactivate or modify any gene in a controlled and efficient manner. To be able to work with the highest technological standards, Zeclinics has licensed this technology from the Broad Institute (MIT/Harvard University) and from ERS genomics.

In some cases, researchers need to screen and validate a long list of candidates to identify the gene responsible for a disease-associated phenotype. During this target validation process, to compare and prioritize different genes, they need a model that allows them to disrupt, with high efficiency, multiple genes in a relatively short time. Thanks to an extremely high performance of the CRISPR/Cas9 system at ZeClinics we are the only zebrafish CRO able to perform a very challenging task: to analyze the effect of gene disruption in only 2-3 months. To obtain this astonishing result, we generate somatic mutants (named CRISPANTS), following few experimental steps:

  1. We adapt the CRISPR/Cas9 system to target our gene(s) of interest;
  2. We inject this customized complex into one-cell-stage zebrafish embryos;
  3. We wait until the embryos reach the appropriate developmental stage;
  4. We analyze injected fish to describe the effect of gene loss-of-function.

If the association between a gene and a pathological condition is already known, the scope of a genetic model can be to help scientists understanding the biological alterations underlying the pathological phenotypes or to screen a library of therapeutic candidates acting against the disease. In this case, the best option is to generate a stable mutant line. To obtain this result, we will start with the procedure described above, designing and injecting a CRISPR/Cas9 system targeting the gene of interest. Injected embryos will be then grown until adulthood, to identify a fish able to transmit a mutation to its progeny thus generating a fish line carrying a mutation in the targeted gene.

Sometimes, it will be needed to test very specific mutations (e.g. single point mutations identified in human patients). In this case, we can use a CRISPR/Cas9-based protocol to insert the desired mutation in the zebrafish genome. This approach is known as knock-in and allows scientists to test the biological effect of specific genetic variants.

The models described so far would be useless if the phenotypes deriving from these genetic manipulations could not be analyzed. Luckily, zebrafish's small size and transparency permit the real-time visualization of an incredibly high number of biological processes. Indeed, thanks to transgenic fish lines expressing fluorescent reporters, it is possible to image organs morphology and activity or to visualize cellular processes like neuronal activation and cell death. In addition, zebrafish larvae are a perfect model for behavioral analysis and allow the study of several pathologies of the nervous system (e.g disorders inducing epileptic seizures, anxiety, or locomotion defects).

This short description points out that zebrafish is an incredible resource that has the potential of supporting the work of scientists from very different backgrounds, ranging from academic researchers interested in basic news and pharmaceutical companies working on the discovery and development of new drugs. At ZeClinics, we take the best out of this model, adapting our genetic services to meet the scientific needs of our clients and to boost their scientific discoveries.

REFERENCES

[1] By De Santis, F., Terriente, J., Di Donato, V. The CRISPR/Cas System in Zebrafish. In Behavioral and Neural Genetics of Zebrafish; Elsevier: Amsterdam, 2020; pp 293–307.

CRISPANTSCRISPR/Cas9drug discoveryGene-editinggenetic modelsgenetic screeningknock-insomatic mutantsZebrafish