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Genetically modifying zebrafish provides more accurate disease models

Genetically modifying zebrafish provides more accurate disease models

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Research published today describes how zebrafish can be genetically engineered to produce disease models that enable more accurate and specific research into a wide variety of human genetic disorders.

CRISPR-Cas9 in action - by Nathan DeveryThe researchers used CRISPR-Cas9 to create the disease models. (Image Credit: Nathan Devery / Shutterstock)

Gene mutations give rise to a wide variety of diseases, including dementia, developmental disorders, some forms of cancer, muscular dystrophies and heart conditions.

Genetic models allow researchers to determine how changes in a particular gene give rise to malfunctions that manifest in a range of disease states.

Due to the complex interactions that may occur between different cell types in a living organism, not all this information can be studied in cell lines.

Zebrafish have become an increasingly popular choice of animal model as large populations can be kept relatively easily and cheaply.

Furthermore, they reach sexual maturity in only 2-3 months and produce up to 300 eggs per week.

Their availability in such large numbers makes them ideal for screening potential new drugs for efficacy and the transparency of their embryos allow non-invasive microscopic investigation of developmental processes.

Advances in gene manipulation techniques have made it possible to readily alter specific gene sequences to observe the effects on normal functioning and on disease states.

In particular, the gene-editing technique CRISPR/Cas9 provides a new level of accuracy and specificity that facilitates in-depth research into human genetic disorders.

It has vastly improved the efficiency with which specific nucleotide changes can be made.

In nature, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a gene-editing technology used by bacterial innate immune system to detect specific regions of the genome using an RNA template.

The enzyme Cas9 is directed to the target where it cleaves the DNA, allowing specific gene sequences to be inserted, deleted, or replaced.

In this way, precise point mutations that replicate those known to cause disease in human patients can be introduced.

CRISPR/Cas9 gene editing is performed on freshly-fertilized single-cell zebrafish embryos so that all cells created as the embryo grows will carry the target mutation.

However, the zebrafish must be screened to confirm which fish carry the desired nucleotide change.

Dr. Lisa Maves, who is studying the involvement of point mutations in congenital heart defects, explained:

There are almost no limitations on what we can design in zebrafish or other systems to generate models of human genetic disorders. CRISPR/Cas9 has really for the first time made it feasible to test the effects of human disease-associated genetic variants in animal models”.

Dr. Lisa Maves, University of Washington School of Medicine

Dr. Andy Willaert and his team at Ghent University are investigating the introduction of errors into the zebrafish genome after CRISPR manipulations to avoid possible misinterpretation of the experimental results obtained after the occurrence of erroneous repair. He commented:

Zebrafish and CRISPR/Cas9 form the ideal duo for massive and swift disease model generation. However, genome editing using a single-stranded repair template often occurs erroneously and commonly used analysis techniques do not always detect such erroneous repair”.

Dr. Andy Willaert, Ghent University

Dr. Federico Tessadori and his team are researching the use of CRISPR/Cas9 technology to develop patient-specific alleles for modeling human cardiovascular disorders caused by point mutations.

Tessadori emphasized “the ability to introduce point mutations exactly replicating the situation in human patients is paramount for proper understanding of disease and for the successful development of therapeutic strategies.”

It is hoped that the enhanced ability to study the basis of human genetic disorders in greater depth will further our understanding of numerous diseases and also identify and screen potential new drug candidates to provide patients with better treatments.

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