Generation of a tgds knock-out zebrafish model for the study of the developmental defects in Catel-Manzke Syndrome

Catel-Manzke syndrome (CMS) is a rare recessive disorder characterized by skeletal and heart malformations due to mutations in the TGDS gene. TGDS is annotated as dTDP-glucose 4,6 dehydratase, because of its homology with the bacterial enzymes of the dTDP-L-rhamnose biosynthesis; however, this pathway was never reported in vertebrates. CMS clinical presentation led to the proposal that TGDS might have a role in glycosaminoglycan formation, but biochemical evidence is lacking and TGDS function remains unknown. Since TGDS is very well conserved during evolution, to address the developmental defects caused by its deficiency, we have developed an F0 zebrafish knock-out model using CRISPR/Cas9. Three sgRNAs targeting different tgds exons were used to form RNP complexes with Cas9. The 3 RNPs mix was injected into the zygotes to obtain F0 biallelic knockouts. Abnormal phenotypes were observed from 24- 48 hpf: they ranged from aberrant head and axial development to less severe skeletal, eye and otic vesicle defects and pericardial edema with venous congestion. These localizations are consistent with the tgds transcript expression determined by ISH. These findings indicate that tgds gene as a role in the early morphogenetic events. No significant phenotypes were seen in the larvae injected with the negative control RNP, thus excluding that the morphological alterations are due to the injection or non-specific Cas9 activity. Genotyping demonstrated that injecting more sgRNA together leads to the formation of large genome deletions between the targeted exons, consistent with previous reports. Mosaicism was observed, as anticipated. Our results suggest that transgenic zebrafish can be a valuable model for delineating tgds function in vertebrates and analyzing the molecular events underlying CMS pathogenesis. We are currently developing a stable line, which will allow more precise morphological, biochemical, and molecular analyses of the phenotypes of the knock-out animals.