347. Tagging FKRP and LARGE by CRISPR/Cas9 for Monitoring Expression and Localization

Abstract

Limb-girdle muscular dystrophy type 2I is caused by mutations in the gene for fukutin-related protein (FKRP). This protein is a putative glycosyltransferase shown to play an important role in the addition of glycans to the muscle membrane protein alpha-dystroglycan (α-DG). The glycosylation of α-DG is accomplished through the function of more than 17 putative and known glycosylatransferases including LARGE, which, when overexpressed, has been shown to compensate for the loss of function in FKRP and Fukutin. However, the exact localization of the endogenous proteins is unknown and no reliable antibody is available for monitoring the changes of their expression. The use of CRISPR/Cas9 for gene editing has been growing in populatiry as a method for introducing mutations into endogenous proteins or as a way of introducing specific sequences, such as a reporter gene. Here we present the use of the CRISPR/Cas9 gene editing system for the addition of a GFP tag to endogenous FKRP and LARGE for the identification of localization as well as to act as a marker for examining changes in expression as a results of treatment with specific compounds. We have applied several techniques to determine the effectiveness of the CRISPR/Cas9 system and encountered many different barriers. The results of these experiments provide valuable information in understanding the role of FKRP in α-DG glycosylation as well as new ways of examining the effects of experimental therapies targeting this glycosylation. With the new knowledge and methods obtained it will be easier to eventually move into in vivo studies to examine their effects in an animal model.