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Published on: April 30, 2018
Generation of desminopathy in rats using CRISPR-Cas9
Henning T Langer1, Agata A Mossakowski1,2, Brandon J Willis3
1Department of Physiology and Membrane Biology, University of California, Davis, CA, USA.
Background:
Desminopathy is a clinically heterogeneous muscle disease caused by over 60 different mutations in desmin. The most common mutation with a clinical phenotype in humans is an exchange of arginine to proline at position 350 of desmin leading to p.R350P. We created the first CRISPR-Cas9 engineered rat model for a muscle disease by mirroring the R350P mutation in humans.
Methods:
Using CRISPR-Cas9 technology, Des c.1045-1046 (AGG > CCG) was introduced into exon 6 of the rat genome causing p.R349P. The genotype of each animal was confirmed via quantitative PCR. Six male rats with a mutation in desmin (n = 6) between the age of 120-150 days and an equal number of wild type littermates (n = 6) were used for experiments. Maximal plantar flexion force was measured in vivo and combined with the collection of muscle weights, immunoblotting, and histological analysis. In addition to the baseline phenotyping, we performed a synergist ablation study in the same animals.
Results:
We found a difference in the number of central nuclei between desmin mutants (1 ± 0.4%) and wild type littermates (0.2 ± 0.1%; P < 0.05). While muscle weights did not differ, we found the levels of many structural proteins to be altered in mutant animals. Dystrophin and syntrophin were increased 54% and 45% in desmin mutants, respectively (P < 0.05). Dysferlin and Annexin A2, proteins associated with membrane repair, were increased two-fold and 32%, respectively, in mutants (P < 0.05). Synergist ablation caused similar increases in muscle weight between mutant and wild type animals, but changes in fibre diameter revealed that fibre hypertrophy in desmin mutants was hampered compared with wild type animals (P < 0.05).
Conclusions:
We created a novel animal model for desminopathy that will be a useful tool in furthering our understanding of the disease. While mutant animals at an age corresponding to a preclinical age in humans show no macroscopic differences, microscopic and molecular changes are already present. Future studies should aim to further decipher those biological changes that precede the clinical progression of disease and test therapeutic approaches to delay disease progression.
Insights
We developed a CRISPR-Cas9 rat model for desminopathy, a muscle disease. Mutant rats showed microscopic and molecular changes, indicating this model
Area of Science:
- Genetics and Molecular Biology
- Muscle Physiology
- Animal Models of Disease
Background:
- Desminopathy is a heterogeneous muscle disorder caused by over 60 mutations in the desmin gene.
- The p.R350P mutation is the most common cause of desminopathy in humans.
- There is a need for robust animal models to study desminopathy pathogenesis and test therapies.
Purpose of the Study:
- To create the first CRISPR-Cas9 engineered rat model for desminopathy.
- To characterize the early molecular and histological changes in this novel model.
- To evaluate the utility of this model for studying disease progression and therapeutic interventions.
Main Methods:
- CRISPR-Cas9 gene editing was used to introduce the desmin mutation (c.1045-1046 AGG > CCG, p.R349P) into the rat genome.
- Genotyping was confirmed using quantitative PCR.
- Phenotypic analysis included in vivo functional tests (maximal plantar flexion force), muscle weight, immunoblotting, histology, and a synergist ablation model.
Main Results:
- Mutant rats exhibited a significant increase in central nuclei within muscle fibers compared to wild-type littermates (P < 0.05).
- Levels of structural proteins including dystrophin, syntrophin, dysferlin, and Annexin A2 were significantly altered in mutant animals (P < 0.05).
- While muscle weight gain was similar after synergist ablation, desmin mutant rats showed hampered fiber hypertrophy compared to wild-type controls (P < 0.05).
Conclusions:
- A novel CRISPR-Cas9 rat model for desminopathy has been successfully generated.
- This model displays preclinical microscopic and molecular alterations, making it valuable for understanding disease progression.
- The model provides a platform for future research into the early biological changes and therapeutic strategies for desminopathy.
