A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish

Abstract

Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

Zebrafish models of sarcopenia

“…Human and zebrafish muscle-related genes have broad functional conservation in muscle growth and development, implying that zebrafish is an excellent model to study human diseases such as myopathies and muscular dystrophies (Gupta et al, 2013). To study human disease-causing variants, researchers have established genetic zebrafish models by utilizing random chemical mutagenesis [using N-ethyl-N-nitrosourea (Henke et al, 2017)] and cutting-edge targeted genomic mutagenesis technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases [TALENs (Radev et al, 2015)] and clustered regularly interspaced short palindromic repeats [CRISPR/Cas9 (Shi et al, 2018)] technology. The establishment of a zebrafish genetic model was primarily based on the molecular and functional characterization of genetic variants presumably associated with muscle phenotypes (Berger et al, 2011).…”

“…In addition, mutation in the human collagen type VI gene (COL6A1 ama605003 ) causes Bethlem myopathy (Box 1) and Ullrich muscular dystrophy (Box 1). The corresponding mutation in zebrafish was the first model used to show collagenassociated muscle myopathy phenotypes, such as muscle fiber disorganization and their folding kinetics (Radev et al, 2015).…”

“…All these phenotypes are usually assessed at embryonic and larval stages. It would be imperative to assess similar muscle phenotypes in adult fish to better understand sarcopenia (Berger et al, 2011;Telfer et al, 2012;Radev et al, 2015). Moreover, currently, no zebrafish genetic models are available to study age-related loss of muscle mass and impaired muscle function.…”

Functional analysis of thyroid hormone receptor beta in Xenopus tropicalis founders using CRISPR-Cas

“…As expected, multiple shorter PCR bands were observed in all embryos injected with the two sgRNAs/Cas9, consistent with these bands being small deletions between the two sgRNA-targeting sites. Injection of both sgRNA/Cas9 RNPs probably induced frameshift mutations and/or small deletions at the exon-intron boundary causing splicing errors ( Radev et al, 2015 ). To examine the possible splicing errors in more detail, we analyzed trβ mRNA from whole bodies of stage 61/62 crispants by RT-PCR using primers designed in the 1st and 3rd exons.…”

Zebrafish Models of Human Disease: Gaining Insight into Human Disease at ZFIN

“…The improvement of reverse genetic techniques such as the use of zinc finger nucleases (Leong et al 2011; Sander et al 2011a, 2011b) CRISPRs (Hruscha and Schmid 2015; Hwang et al 2013; Hwang et al 2015) and transcription activator-like effector nucleases (TALENs; Huang et al 2011, 2016; Ma et al 2016; Sander et al 2011a, 2011b) have allowed for the targeted mutation of disease-specific genes. These techniques are becoming the method of choice in zebrafish for creating genetic models of human disease, such as models for visceral heterotaxy, Bethlem myopathy, and Allan-Herndon-Dudley syndrome (Table 1A; Noël et al 2015; Radev et al 2015; Zada et al 2014). …”