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A Transient Expression System with Stably Integrated CRISPR-dCas9 Fusions for Regulation of Genes Involved in Immunoglobulin G Glycosylation.

Author information

Affiliations

  • 1. Department of Biology, Division of Molecular Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia; University of Zagreb, Zagreb, Croatia.
      Authors
    • Mijakovac A 1
    • Miškec K 1
    • Vičić Bočkor V 1
    • Zoldoš V 1
    • Vojta A 1
    (5 authors)
  • 2. Genos Glycoscience Research Laboratory, Zagreb, Croatia; University of Zagreb, Zagreb, Croatia.
      Authors
    • Krištić J 2
    • Lauc G 2
    (2 authors)
  • 3. Division of Molecular Biology, Laboratory for Cell Biology and Signaling, Ruđer Bošković Institute, Zagreb, Croatia; University of Zagreb, Zagreb, Croatia.
      Authors
    • Tadić V 3
    (1 author)
  • 4. Laboratory for Cancer research, University of Split School of Medicine, Split, Croatia; and University of Zagreb, Zagreb, Croatia.
      Authors
    • Bošković M 4
    (1 author)

ORCIDs linked to this article

The CRISPR Journal, 12 Jan 2022, 5(2):237-253
https://doi.org/10.1089/crispr.2021.0089 PMID: 35021898 

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.

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Read article at publisher's site: https://doi.org/10.1089/crispr.2021.0089

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