First Characterization of Human Dermal Fibroblasts Showing a Decreased Xylosyltransferase-I Expression Induced by the CRISPR/Cas9 System

Abstract

Background: Xylosyltransferases-I and II (XT-I and XT-II) catalyze the initial and rate limiting step of the proteoglycan (PG) biosynthesis and therefore have an import impact on the homeostasis of the extracellular matrix (ECM). The reason for the occurrence of two XT-isoforms in all higher organisms remains unknown and targeted genome-editing strategies could shed light on this issue. Methods: XT-I deficient neonatal normal human dermal fibroblasts were generated by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated proteins (Cas) 9 system. We analyzed if a reduced XT-I activity leads to abnormalities regarding ECM-composition, myofibroblast differentiation, cellular senescence and skeletal and cartilage tissue homeostasis. Results: We successfully introduced compound heterozygous deletions within exon 9 of the XYLT1 gene. Beside XYLT1, we detected altered gene-expression levels of further, inter alia ECM-related, genes. Our data further reveal a dramatically reduced XT-I protein activity. Abnormal myofibroblast-differentiation was demonstrated by elevated alpha-smooth muscle actin expression on both, mRNA- and protein level. In addition, wound-healing capability was slightly delayed. Furthermore, we observed an increased cellular-senescence of knockout cells and an altered expression of target genes knowing to be involved in skeletonization. Conclusion: Our data show the tremendous relevance of the XT-I isoform concerning myofibroblast-differentiation and ECM-homeostasis as well as the pathophysiology of skeletal disorders.