Abstract
Aberrant fucosylation in cancer cells is considered as a signature of malignant cell transformation and 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 the deregulated expression of pertinent fucosyltransferases, like fucosyltransferase 4 (FUT4) and fucosyltransferase 9 (FUT9). However, the lack of experimental models closely mimicking cancer-specific regulation of fucosyltransferase gene expression has, so far, limited our knowledge regarding the substrate specificity of these enzymes and the impact of Lewisx synthesis on the glycome of colorectal cancer cells. Therefore, we sought to transcriptionally activate the Fut4 and Fut9 genes in the well-known murine colorectal cancer cell line, MC38, which lacks expression of the FUT4 and FUT9 enzymes. For this purpose, we utilized a physiologically relevant, guide RNA-based model of de novo gene expression, namely the CRISPR-dCas9-VPR system. Induction of the Fut4 and Fut9 genes in MC38 cells using CRISPR-dCas9-VPR resulted in specific neo-expression of functional Lewisx antigen on the cell surface. Interestingly, Lewisx was mainly carried by N-linked glycans in both MC38-FUT4 and MC38-FUT9 cells, despite pronounced differences in the biosynthetic properties and the expression stability of the induced enzymes. Moreover, Lewisx expression was found to influence core-fucosylation, sialylation, antennarity and the subtypes of N-glycans in the MC38-glycovariants. In conclusion, exploiting the CRISPR-dCas9-VPR system to augment glycosyltransferase expression is a promising method of transcriptional gene activation with broad application possibilities in glycobiology and oncology research.
Highlights
Over the last few years, genome engineering through the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has received increased attention and is gradually becoming a method of choice for studying various biological processes, including protein and lipid glycosylation
A key factor for precise, and efficient, gene targeting using the CRISPR-dCas9-VPR system is the design of the corresponding guide RNA (gRNA) sequences
We initially reviewed the literature giving emphasis to the promoter regions as well as the known regulatory elements that have been implicated in determining the expression of the fucosyltransferase 4 gene (Fut4) and fucosyltransferase 9 gene (Fut9) fucosyltransferase genes
Summary
Over the last few years, genome engineering through the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has received increased attention and is gradually becoming a method of choice for studying various biological processes, including protein and lipid glycosylation. In sharp contrast with traditional methods of gene overexpression with the use of selected cDNA clones, the CRISPR-dCas9-VPR toolkit facilitates de novo gene transcription that occurs physiologically within the nucleus of the cell and its native chromosomal context. In this case, one or multiple guide RNA (gRNA) sequences target the promoter region of the gene of interest, resulting in direct recruitment of the catalytically inactive Cas nuclease (called defective or deactivated Cas9) to this site. A major difference compared to the CRISPR-Cas gene editing tools is that the dCas protein is fused to a hybrid tripartite activation domain (VP64-p53-Rta), known as VPR. The subsequent interaction between the VPR activation unit of dCas and the RNA polymerase II and/or other transcription factors eventually drives the expression of the gene of interest (Figure 1A)
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