In addition, MAL-I binding to MC38-FUT4 and MC38-FUT9 cells could be rescued upon treatment of the cells with 2F-PF. Open in a separate window Fig. In this case, one or multiple guideline RNA (gRNA) sequences specifically target the promoter region of the gene of interest, resulting in direct recruitment of the catalytically inactive Cas9 nuclease (called defective or deactivated Cas9) to this site. However, a major difference compared to the CRISPR-Cas9 gene editing tools is that the dCas9 protein is now fused to a hybrid tripartite activation domain name (VP64-p53-Rta), known as VPR. The subsequent interaction between the VPR activation unit of dCas9 and the RNA polymerase II and/or other transcription factors eventually drives the expression of the gene of interest (Physique ?(Figure11A). Open in a separate windows Fig. 1. Model and experimental design for the CRISPR-dCas9-VPR system. (A) Theory of transcriptional gene activation using the CRISPR-dCas9-VPR technology. One or multiple guideline RNA (gRNA) sequences that specifically target the complementary promoter region of the (glyco)gene of interest, result in direct recruitment of the catalytically inactive Cas9 nuclease (known as defective or deactivated Cas9) to this site. The following conversation between VPR (VP64-p65-Rta chimeric activator fused to the C-terminus of dCas9) and RNA polymerase II drives the induction of target gene expression. (B) Overview of the five-step experimental design applied for transcriptional activation of the murine and genes using the CRISPR-dCas9-VPR technology. We hypothesized that induction of gene expression using the CRISPR-dCas9-VPR system could be reliably applied to glycobiology research through the efficient and specific transcriptional programming of glycosyltransferase genes. Importantly, by employing CRISPR-dCas9-VPR, all the critical regulatory mechanisms associated with glycosyltransferase gene expression can be very easily unraveled, since they are still active in this model and not simply bypassed. In the past, significant changes in glycosylation due to the use of cDNA clones have been observed (van Leeuwen et al. 2006). Moreover, complex epigenetic modifications of genes involved in protein and lipid glycosylation (Zoldos et al. 2010, Lauc et al. 2014) that are often completely missed or undermined when cDNA clones are used, can be now further assessed with CRISPR-dCas9-VPR (Lo and Qi 2017). This is of utmost importance for dissecting the mechanisms that lead to an aberrant expression profile of certain glycosyltransferases under pathological conditions, as in the case of cancer. In general, tumor cells are characterized by a tremendous switch in their cell surface glycome, as a result of genetic or epigenetic alterations in the expression of particular glycosyltransferase genes. Specifically, malignancy cells exhibit elevated levels of fucosylation, sialylation and branched and fucosyltransferase genes in MC38 cells, a murine colorectal adenocarcinoma cell collection that is generally used in pre-clinical mouse models for this disease (McIntyre et al. 2015, Zhao et al. 2017). Following this approach, we successfully generated FUT4- or FUT9-expressing MC38 glyco-engineered cell lines and examined changes in their respective glycosylation profiles, focusing on biosynthesis of the fucosylated Lewisx determinant and its impact on the malignancy cell glycome. We believe that this novel methodology of gene expression can be further applied both to human and murine glycosyltransferases involved in tumorigenesis or other disorders and thus set the framework to elucidate the exact implication of these enzymes (or their synthesized glycan structures) in different aspects of BKM120 (NVP-BKM120, Buparlisib) disease pathogenesis. Moreover, we consider our study as a representative example of how improvements in the CRISPR technology can benefit research investigations focused on glycosylation, thus highlighting its role in health and disease. Results Design, selection and quality control of the murine and gene targeting gRNA sequences A key factor for precise, but also efficient, gene targeting using the CRISPR-dCas9-VPR system is the design of the corresponding gRNA sequences. To date, several prediction tools have been developed for this purpose (Hsu et al. 2013, Doench et al. 2014, Heigwer et al. 2014, Montague et al. 2014), providing detailed lists of proposed gRNAs to the user. However, the final decision about the exact gRNAs that should be used remains a big challenge and a protocol for precisely narrowing down all the possible options is still missing. Therefore, we here present the.Therefore, we here present the workflow followed by us for the selection and quality control of the designed gRNAs targeting the murine and genes (Figure ?(Figure1B).1B). 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 and genes in MC38 cells using CRISPR-dCas9-VPR resulted in specific neo-expression of functional Lewisx antigen around the cell surface. Interestingly, Lewisx was mainly carried by gene transcription that occurs physiologically within the nucleus of the cell and its native chromosomal context. In this case, one or multiple guideline RNA (gRNA) sequences specifically target the promoter region of the gene of interest, resulting in direct BKM120 (NVP-BKM120, Buparlisib) recruitment of the catalytically inactive Cas9 nuclease (called defective or deactivated Cas9) to this site. However, a major difference compared to the CRISPR-Cas9 gene editing tools is that the dCas9 protein is now fused to a hybrid tripartite activation domain name (VP64-p53-Rta), known as VPR. The subsequent interaction between the VPR activation unit of dCas9 and the RNA polymerase II and/or other transcription factors eventually drives the expression of the gene of interest (Physique ?(Figure11A). Open in a separate windows Fig. 1. Model and experimental design for the CRISPR-dCas9-VPR system. (A) Theory of transcriptional gene activation using the CRISPR-dCas9-VPR technology. One or multiple guideline RNA (gRNA) sequences that specifically target the complementary promoter region of the (glyco)gene of interest, result in direct recruitment of the catalytically inactive Cas9 nuclease (known as defective or deactivated Cas9) to this site. The following conversation between VPR (VP64-p65-Rta chimeric activator fused to the C-terminus of dCas9) and RNA polymerase II drives the induction of target gene expression. (B) Overview of the five-step experimental design applied for transcriptional activation of the murine and genes using the CRISPR-dCas9-VPR technology. We hypothesized that induction of gene expression using the CRISPR-dCas9-VPR system could be reliably applied to glycobiology research through the efficient and specific transcriptional programming of glycosyltransferase genes. Importantly, by employing CRISPR-dCas9-VPR, all the critical regulatory mechanisms associated with glycosyltransferase gene expression can be very easily unraveled, since they are still active in this model and not simply bypassed. In the past, significant changes in glycosylation due to the use of cDNA clones have been observed (van Leeuwen et al. 2006). Rabbit Polyclonal to iNOS (phospho-Tyr151) Moreover, complex epigenetic modifications of genes involved in protein and lipid glycosylation (Zoldos et al. 2010, Lauc et al. 2014) that are often completely missed or undermined when cDNA clones are used, can be now further assessed with CRISPR-dCas9-VPR (Lo and Qi 2017). This is of utmost importance for dissecting the mechanisms that lead to an aberrant expression profile of certain glycosyltransferases under pathological conditions, as in the case of cancer. In general, tumor cells are characterized by a tremendous switch in their cell surface glycome, as a result of genetic or epigenetic alterations in the expression of particular glycosyltransferase genes. Specifically, cancer cells exhibit elevated levels of fucosylation, sialylation and branched and fucosyltransferase genes in MC38 cells, a murine colorectal adenocarcinoma cell collection that is commonly used in pre-clinical mouse models for this disease (McIntyre et al. 2015, Zhao et al. 2017). Following this approach, we successfully generated FUT4- or FUT9-expressing MC38 glyco-engineered cell lines and examined changes in their respective glycosylation profiles, focusing on biosynthesis of the fucosylated Lewisx determinant and its BKM120 (NVP-BKM120, Buparlisib) impact on the cancer cell glycome. We believe that this novel methodology of gene expression can be further applied both to human and murine glycosyltransferases involved in tumorigenesis or other disorders and thus set the framework to elucidate the exact implication of these enzymes (or their synthesized glycan structures) in different aspects of disease pathogenesis. Moreover, we consider our study as a representative example of how advances in the CRISPR technology can benefit research investigations focused on glycosylation, thus highlighting its role in health and disease. Results Design, selection and quality control of the murine and gene targeting gRNA sequences A key factor for precise, but also efficient, gene targeting using the CRISPR-dCas9-VPR system is the design of the corresponding gRNA sequences. To date, several prediction tools have been developed for this purpose (Hsu et al. 2013, Doench et al. 2014, Heigwer et al. 2014, Montague et al. 2014), providing detailed lists of proposed gRNAs to the user. However, the final decision about the exact gRNAs that should be used remains a big challenge and a protocol for precisely narrowing down all the possible options is still missing. Therefore, we here present the workflow followed by us for the selection and quality control of the designed gRNAs targeting the murine and genes (Figure ?(Figure1B).1B). Importantly, we believe that this process can be easily adapted and utilized for the selection of gRNA sequences that specifically target any other (glyco)gene of interest. In.