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

Mijakovac, Anika; Miškec, Karlo; Krištić, Jasminka; Bočkor, Vedrana Vičić; Tadić, Vanja; Bošković, Maria; Lauc, Gordan; Zoldoš, Vlatka; Vojta, Aleksandar

doi:10.1089/crispr.2021.0089

Cited by 8 publications

(17 citation statements)

References 44 publications

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“…A recent GWAS discovered a gene network involving 27 loci implicated in the process of IgG glycosylation with most of them associated with various autoimmune and inflammatory conditions ( Klarić et al, 2020 ). Furthermore, part of this gene network was functionally validated in vitro utilizing a recently developed transient expression system based on CRISPR tools ( Mijakovac et al, 2021 , 2022 ). Despite this tight genetic control of the IgG glycome that is currently being unraveled, heritability analysis of the glycan clock revealed only a moderate genetic contribution averaging around 39% for both cohorts.…”

Section: Discussionmentioning

confidence: 99%

Heritability of the glycan clock of biological age

Mijakovac

Frkatović²,

Ivok³

et al. 2022

Front. Cell Dev. Biol.

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Immunoglobulin G is posttranslationally modified by the addition of complex N-glycans affecting its function and mediating inflammation at multiple levels. IgG glycome composition changes with age and health in a predictive pattern, presumably due to inflammaging. As a result, a novel biological aging biomarker, glycan clock of age, was developed. Glycan clock of age is the first of biological aging clocks for which multiple studies showed a possibility of clock reversal even with simple lifestyle interventions. However, none of the previous studies determined to which extent the glycan clock can be turned, and how much is fixed by genetic predisposition. To determine the contribution of genetic and environmental factors to phenotypic variation of the glycan clock, we performed heritability analysis on two TwinsUK female cohorts. IgG glycans from monozygotic and dizygotic twin pairs were analyzed by UHPLC and glycan age was calculated using the glycan clock. In order to determine additive genetic, shared, and unique environmental contributions, a classical twin design was applied. Heritability of the glycan clock was calculated for participants of one cross-sectional and one longitudinal cohort with three time points to assess the reliability of measurements. Heritability estimate for the glycan clock was 39% on average, suggesting a moderate contribution of additive genetic factors (A) to glycan clock variation. Remarkably, heritability estimates remained approximately the same in all time points of the longitudinal study, even though IgG glycome composition changed substantially. Most environmental contributions came from shared environmental factors (C), with unique environmental factors (E) having a minor role. Interestingly, heritability estimates nearly doubled, to an average of 71%, when we included age as a covariant. This intervention also inflated the estimates of unique environmental factors contributing to glycan clock variation. A complex interplay between genetic and environmental factors defines alternative IgG glycosylation during aging and, consequently, dictates the glycan clock’s ticking. Apparently, environmental factors (including lifestyle choices) have a strong impact on the biological age measured with the glycan clock, which additionally clarifies why this aging clock is one of the most potent biomarkers of biological aging.

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Section: Discussionmentioning

confidence: 99%

Heritability of the glycan clock of biological age

Mijakovac

Frkatović²,

Ivok³

et al. 2022

Front. Cell Dev. Biol.

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“…Given that the majority of identified candidate SNPs are located in non-coding regions, we applied several in silico approaches to prioritize potential causal genes in associated genomic regions, including exploring functional consequences of associated variants, pleiotropy with gene expression, gene-based association analysis, as well as evidence from previous GWA studies and prior biological knowledge of gene function. Among 37 candidate genes from 16 associated loci, for the two genes, RUNX3 and B4GALT1, the effect on IgG glycosylation has recently been demonstrated using direct gene expression modulation in HEK293FS IgG transient expression system 32,40 .…”

Section: Discussionmentioning

confidence: 99%

“…The functional follow-up was based on the previously developed HEK293FS transient expression system for IgG production which was shown to be an excellent tool for studying the role of the genes identified in glycosylation GWAS 32 . Despite the fact that we could not demonstrate upregulation of HIVEP2 mRNA using dCas9-VPR, a significant increase in digalactosylated structures with a concomitant decrease in agalactosylated structures was measured from IgG secreted from the same cells.…”

Section: Discussionmentioning

confidence: 99%

“…To functionally validate the potential role of the selected GWAS hits in IgG galactosylation, we used the newly developed transient expression system HEK293FS for IgG production with stably integrated dSaCas9-VPR or dSpCas9-KRAB expression cassettes for target gene upregulation or downregulation 32 . Therefore, the candidate genes were manipulated in dCas9-VPR or dCas9-KRAB containing monoclonal cell lines transiently cotransfected with a plasmid carrying genes for IgG heavy and light chains and specific gRNA.…”

Section: Functional Validation Of Gwas Hitsmentioning

confidence: 99%

“…Here, we conducted a GWAS of IgG galactosylation phenotypes in a study that almost doubles the sample size (N=13,705) compared to previous GWAS of IgG N-glycome 30 and focus on the genes with in-silico evidence for involvement in the IgG galactosylation process. To assess their functional role in IgG galactosylation we applied our recently developed HEK293FS IgG transient expression system based on CRISPR-dCas9 molecular tools to functionally test whether the change in the expression of associated genes affects the IgG galactosylation levels 32 .…”

Section: Introductionmentioning

confidence: 99%

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Mapping of the gene network that regulates glycan clock of ageing

Frkatović

Miškec

Mijakovac

et al. 2023

Preprint

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Glycans are an essential structural component of Immunoglobulin G (IgG) that modulate its structure and function. However, regulatory mechanisms behind this complex posttranslational modification are not well known. Previous genome-wide association studies (GWAS) identified 29 genomic regions involved in regulation of IgG glycosylation, but only a few were functionally validated. One of the key functional features of IgG glycosylation is the addition of galactose (galactosylation). We performed GWAS of IgG galactosylation (N=13,705) and identified 16 significantly associated loci, indicating that IgG galactosylation is regulated by a complex network of genes that extends beyond the galactosyltransferase enzyme that adds galactose to IgG glycans. Gene prioritization identified 37 candidate genes. Using a recently developed CRISPR/dCas9 system we manipulated gene expression of candidate genes in thein vitroIgG expression system. Up- and downregulation of three genes,EEF1A1,MANBAandTNFRSF13B, changed the IgG glycome composition, which confirmed that these three genes are involved in IgG galactosylation in this in vitro expression system.

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