| Progressive muscular dystrophy is a group of muscular degeneration caused by genetic defects,characterized by degeneration and necrosis of skeletal muscle fibers,which slowed progressive muscle atrophy and muscle weakness in clinical and also involved the heart and skeletal system.According to the different clinical manifestations and mutation genes,it can be divided into Duchenne muscular dystrophy(DMD)and Limb girdle muscular dystrophy.Duchenne muscular dystrophy is an X-linked muscle-wasting disorder caused by mutations in the dystrophin gene,with an incidence of 1 in 3500 in new male births.Mdx mice are widely used as an animal model for DMD.However,the phenotype of mdx mice is much milder as compared to DMD patients with almost a normal life span,which is not faithful recapitulate DMD patients and used to the safety assessment for clinical drug screening.Although larger animal models of DMD such as dogs and pigs have been generated,the usage of these animals is expensive and only limited in several facilities in the world.Limb girdle muscular dystrophy type-2L(LGMD2L)is autosomal recessive muscular dystrophy caused by mutations in the gene encoding anoctamin-5(ANO5),which belongs to the anoctamin protein family.Two independent lines of mice with complete disruption of ANO5 transcripts did not exhibit overt muscular dystrophy phenotypes;instead,one of these mice was observed to present with some abnormality in sperm motility.In contrast,a third line of ANO5-knockout(KO)mice with residual expression of truncated ANO5 expression was reported to display defective membrane repair and very mild muscle pathology.At present,it is unclear the reason for the different of pathological phenotypes in ANO5 knockout mice.Therefore,it is necessary to develop new species animal models for related research.At present,there is a lack of effective treatment measures and appropriate animal models for these diseases.Therefore,it is urgent to establish a better animal model to simulate muscular dystrophy and screen effective drugs and treatment methods.The rabbit is a new species animal model,which has more similarities with human beings in terms of physiology,anatomy and genetics than mice and rat,and also require short pregnancy period,low cost maintenance and easy to operate compared to the pigs and monkeys.The rabbit as animal models of muscular dystrophy is still blank in the world.Therefore,the construction of rabbit disease model will provide a reliable and ideal animal model for better simulation of human genetic diseases.Here we report the generation of a rabbit model of DMD by co-injection of Cas9 mRNA and sgRNA targeting exon 51 into rabbit zygotes.The DMD-knockout(KO)rabbits exhibit the typical phenotypes of DMD,including severely impaired physical activity,elevated serum creatine kinase levels,and progressive muscle necrosis and fibrosis.Moreover,clear pathology was also observed in the diaphragm and heart at 5 months of age,similar to DMD patients.Echocardiography recording showed that the DMD-KO rabbits had chamber dilation with decreased ejection fraction and fraction shortening.In conclusion,this novel rabbit DMD model generated with the CRISPR/Cas9 system mimics the histopathological and functional defects in DMD patients,and make up for the shortcomings of other animal models.Up to now,many of the ANO5-related patients carry point mutations or small insertions/deletions(indels)in the ANO5 gene.To more closely mimic the human ANO5 mutations,we engineered mutant ANO5 rabbits with small indels in the exon 12 and/or 13.The mutant rabbit showed no pathological changes at birth and the development of typical signs of muscular dystrophy at at 12 months of age,including increased serum creatine kinase(CK),muscle necrosis,regeneration,fatty replacement and fibrosis,the features of age-dependent are similar to clinical patients This novel ANO5 mutant rabbit model would be useful in studying the disease pathogenesis and therapeutic treatments for ANO5-deficient muscular dystrophy.In addition,we performed the exon skipping of DMD and ANO5 gene-edited rabbits,and found the existed of exon skipping in rabbits.In order to further explore the mechanism of exon skipping,by analysing 22 gene-edited rabbit lines to study the patterns of exon skipping,revealed that premature termination codon mutation can lead to exon skipping.In summary,we generated a novel rabbit model with DMD or ANO5 mutations,compared to other animal model,which is well recapitulate human DMD and LGMD patients,and provide an ideal animal disease model for clinical drug screening and the study of pathological mechanisms,meanwhile,the premature termination codon mutations(PTC)is a main factor to leading to CRISPR/Cas9-mediated exon skipping. |
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