Transcriptional activation of fucosyltransferase (FUT) genes using the CRISPR-dCas9-VPR technology reveals potent N-glycome alterations in colorectal cancer cells

Aberrant fucosylation in cancer cells is considered as a signature of malignant cell transformationand it is associated with tumor progression, metastasis and resistance to chemotherapy. Specifically,in colorectal cancer cells, increased levels of the fucosylated Lewisx antigen are attributed to thederegulated expression of pertinent fucosyltransferases, like fucosyltransferase 4 (FUT4) and fucosyltransferase9 (FUT9). However, the lack of experimental models closely mimicking cancer-specificregulation of fucosyltransferase gene expression has, so far, limited our knowledge regarding thesubstrate specificity of these enzymes and the impact of Lewisx synthesis on the glycome of colorectalcancer cells. Therefore, we sought to transcriptionally activate the Fut4 and Fut9 genes in the wellknownmurine colorectal cancer cell line, MC38, which lacks expression of the FUT4 and FUT9enzymes. For this purpose, we utilized a physiologically relevant, guide RNA-based model of de novogene expression, namely the CRISPR-dCas9-VPR system. Induction of the Fut4 and Fut9 genes inMC38 cells using CRISPR-dCas9-VPR resulted in specific neo-expression of functional Lewisx antigenon the cell surface. Interestingly, Lewisx was mainly carried by N-linked glycans in both MC38-FUT4and MC38-FUT9 cells, despite pronounced differences in the biosynthetic properties and the expressionstability of the induced enzymes. Moreover, Lewisx expression was found to influence corefucosylation,sialylation, antennarity and the subtypes of N-glycans in the MC38-glycovariants. Inconclusion, exploiting the CRISPR-dCas9-VPR system to augment glycosyltransferase expression isa promising method of transcriptional gene activation with broad application possibilities in glycobiologyand oncology research.