P.248CRISPR-Cas9 tagging allows the detection of endogenous gne in mice

Abstract

GNE myopathy is an autosomal recessive adult onset disorder caused mainly by missense mutations in the GNE gene. Although GNE function in the biosynthesis pathway of sialic acid is well established, the mechanism leading from GNE mutation to this myopathy is unclear. In an attempt to elucidate GNE functions that could account for the muscle pathophysiology of this disorder, it is crucial to monitor GNE expression, tissue distribution and subcellular localization that might shed some light on GNE additional function(s) in muscle. To date, no reliable antibodies can detect the endogenous levels of the GNE protein. Therefore we have targeted the mouse endogenous Gne locus with two different independent tags to monitor Gne's specific expression and subcellular localization. We used the crRNA:tracrRNA Crispr/Cas complex strategy to engineer the carboxi-terminus of the Gne gene and introduced either a 3X FLAG or a 3X HA tag in mouse zygotes using a ssDNA donor for homologous recombination. The resulting mice were analyzed by western blot with specific antibody for each tag. A Gne product of ∼80 kDa, as expected, was detected in the various tissues examined. The endogenous Gne was successfully detected even in tissues with low expression level, like skeletal muscles. BothGne alleles were found to be expressed, resulting in a 2-fold increase in protein detection in homozygous versus heterozygous Gne tagged mice. These Gne-tagged mice will be a useful platform for the follow up of the spatio-temporal expression of Gne, for comparison between wild type and mutant protein expression and for the establishment of a GNE myopathy model. Further, it can be used as the basis of a conditional knock-out mice that might shed some light about the Gne mechanisms leading to the disease in muscle.