CRISPR-based epigenome and genome editing reveal novel roles for PLEC in cartilage biology, mechanobiology, and response to inflammation

<title>Abstract</title> <p> <bold>Background</bold> Osteoarthritis (OA)-associated single nucleotide polymorphism (SNP) rs11780978 has previously been associated with differential methylation at sites within <italic>PLEC</italic> gene and differential expression of <italic>PLEC</italic> in multiple joint tissues, including cartilage. We undertook experiments to identify novel differential methylation sites and used epigenome editing to link rs11780978 associated differential methylation to <italic>PLEC</italic> expression <italic>.</italic> Here, we established a novel pipeline using immortalized human chondrogenic cells to determine the role of <italic>PLEC.</italic> CRISPR based genome editing was employed to replicate plectin deficiency followed by deep phenotyping through cartilage development (chondrogenesis), and homeostasis. <bold>Methods</bold> Methylation quantitative trait loci (mQTL) analyses of novel CpGs were undertaken. Epigenome editing was employed to modulate methylation at CpGs of interest and expression of genes of interest was screened by qPCR. A novel, highly chondrogenic CRISPR based cell line was used to develop a <italic>PLEC</italic> -knockout and CTRL line. Cells underwent chondrogenesis and were phenotyped by qPCR as well as staining/immunohistochemistry for matrix components. Cells were challenged with mechanical and mechano-inflammatory stimuli and phenotyped by high depth RNA sequencing. <bold>Results</bold> Two previously described and four novel mQTLs were replicated in human OA cartilage. The six mQTLs were targeted with epigenome modulation which prioritised <italic>PLEC</italic> for further study. Plectin editing was efficient at the DNA, mRNA and protein level and was maintained throughout chondrogenesis and mechano-inflammatory challenge. Plectin deficiency was associated with molecular phenotypes, including reducing anabolism following chondrogenesis. Deep phenotyping of the chondrocyte response to loading and combined mechano-inflammation identified genes and pathways of interest intersecting with plectin deficiency. <bold>Conclusions</bold> We report a functional pipeline to test a non-coding OA SNP from epigenome editing, through genome editing and downstream deep phenotypic characterisation of genes of interest in in vitro cartilaginous model systems replicating the mechano-inflammatory environment. This system enabled us to identify multiple novel links between the functional region, plectin and chondrocyte biology and mechanobiology. These data enhance our knowledge on plectin biology and provide new pathways of interest for future study. </p>