A Transient Expression System with Stably Integrated CRISPR-dCas9 Fusions for Regulation of Genes Involved in Immunoglobulin G Glycosylation
Anika Mijakovac, Karlo Miškec, Jasminka Krištić, Vedrana Vičić Bočkor, Vanja Tadić, Maria Bošković, Gordan Lauc, Vlatka Zoldoš, Aleksandar Vojta
Abstract
Alternative glycosylation of immunoglobulin G (IgG) is functionally important in multiple human physiological and pathological states. Our understanding of molecular mechanisms that regulate IgG glycosylation is vague because of the complexity of this process, which involves hundreds of genes. Several genome-wide association (GWA) studies have revealed a network of genes associated with IgG glycosylation that are pleiotropic for a number of diseases. Here, we report a design of a versatile system for IgG production and gene manipulations that can be used for in vitro functional follow-up of GWA hits or any gene of interest. The system is based on CRISPR-dCas9, extended by a piggyBac integrase compatible vector, and drives IgG production in HEK-293F cells. We validated our systems that stably express VPR-dCas9 and KRAB-dCas9 by manipulation of four glyco-genes with a known role in IgG glycosylation, and then functionally validated three GWAS hits for IgG glycosylation with an as-yet-unknown role in this process.