Abstract
Limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi myopathy type 3 (MMD3) are 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. 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 via co-injection of Cas9 mRNA and sgRNA into the zygotes. CRISPR-mediated small indels in the exon 12 and/or 13 in the mutant rabbits lead to the development of typical signs of muscular dystrophy with increased serum creatine kinase (CK), muscle necrosis, regeneration, fatty replacement and fibrosis. This novel ANO5 mutant rabbit model would be useful in studying the disease pathogenesis and therapeutic treatments for ANO5-deficient muscular dystrophy.
Highlights
The limb girdle muscular dystrophies (LGMDs) are a diverse group of childhood and adult onset muscle diseases, characterized by progressive weakness of the hip and shoulder girdles and of the lower limbs, with muscle atrophy
We showed that engineered small insertions/deletions in the ANO5 gene of rabbits lead to muscle degeneration/regeneration, atrophy and elevation of serum creatine kinase (CK) levels
These results showed that the dual single guide RNA (sgRNA)-directed CRISPR/Cas[9] system is efficient for generation of mutations in the ANO5 gene in rabbit embryos
Summary
To generate an ANO5-KO model, we designed a pair of sgRNAs targeting exon 12 and exon 13 to disrupt the open reading frame (ORF) of ANO5 in rabbit (Fig. 1a). There were no significant differences in the developmental rate between the non-injected embryos and microinjected embryos (p > 0.05) These results showed that the dual sgRNA-directed CRISPR/Cas[9] system is efficient for generation of mutations in the ANO5 gene in rabbit embryos. ANO5 rabbits displayed increased percentage of centrally nucleated fibers and necrotic area (Fig. 3c, d) The appearance of these phenotypes was typically observed starting at 12 months of age. The bladder of the ANO5-/- rabbits displayed extensive fibrosis These studies suggest that the loss-of-function mutations in the ANO5 gene lead to the pathological changes in both skeletal and smooth muscles. At 14 days after cardiotoxin injection, the gastrocnemius muscle from the wild-type rabbits showed typical signs of regeneration as evidenced by the presence of central nuclei (Fig. 6a). These results suggest that ANO5 plays a role in muscle regeneration
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
