Naslov
Regulation of genes involved in IgG glycosylation using a transient expression system with stably integrated CRISPR/dCas9 fusions

Autori
Aleksandar Vojta, Anika Mijakovac, Karlo Miškec, Jasminka Krištić, Vedrana Vičić Bočkor, Gordan Lauc, Vlatka Zoldoš

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, ostalo

Izvornik
Glycobiology / - , 2021, 1713-1713

Skup
2021 ANNUAL MEETING OF THE SOCIETY FOR GLYCOBIOLOGY

Mjesto i datum
San Diego (CA), Sjedinjene Američke Države, 07.11.2021. - 10.11.2021

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
IgG, CRISPR/dCas9, glycosylation

Sažetak
Alternative glycosylation of immunoglobulin G (IgG) is functionally important in multiple human physiological and pathological states. One of the best examples is a simple fucosylation that can switch IgG effector function between pro- and anti- inflammatory, which is critical in production of therapeutic antibodies. Several genome-wide association studies revealed a network of genes associated with IgG glycosylation which are pleiotropic for several diseases. To understand the complex molecular mechanisms that regulate IgG glycosylation, we designed a versatile system for IgG production and gene manipulation 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, essentially creating derived cell lines with integrated machinery for programmed transcriptional control. Transient transfection of those cells with the second component of the system, a plasmid for monocistronic expression of both IgG chains and co-expression of gRNA molecules, enables the readout of IgG glycan profiles following precisely targeted up- or downregulation of genes of interest. Though the development of the system was complex, its utilization by the end user is simple and straightforward. Transfection of the large dCas9 fusion constructs is quite inefficient and represents a bottleneck in effective experimental design with high throughput. The bipartite nature of the system ensures that only the cells expressing gRNA (with concomitant transcriptional alteration) produce IgG that is analyzed for glycosylation profile. We validated our derived cell lines that stably express VPR-dCas9 and KRAB-dCas9 by manipulation of four glycogenes with a known role in IgG glycosylation. Transcriptional up- and downregulation of B4GALT1, encoding the enzyme that adds galactose to the core glycan structure, had the predictable effect on the abundance of agalactosylated and galactosylated structures, changing the glycosylation profile depending on the transcript level. Similarly, downregulation of FUT8, encoding a fucosyltransferase, decreased the core-fucosylated structures, while upregulation of ST6GAL1 and MGAT3 increased sialylated structures and those with a bisecting GlcNAc, respectively, which was in line with the expected change in enzyme levels. Interestingly, the change in sialylated structures was accompanied by changes in galactosylation, again demonstrating the complexity of the IgG glycosylation network. Our newly developed system for transient expression of can be used for studying the role of genes associated with IgG glycosylation by GWA studies. It can also be easily repurposed to serve as a model for other proteins and their posttranslational modifications, with appropriate targeting via gRNA.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Biotehnologija u biomedicini (prirodno područje, biomedicina i zdravstvo, biotehničko područje)

POVEZANOST RADA