Molecular tools for the glycosciences
CRISPR Cas-9 Tools
O-linked glycans, N-linked glycans and glycosphingolipids (GSLs) are three major glycoconjugate-types on the mammalian cell surface. The following are CRISPR-Cas9 plasmid vectors available from Addgene to truncate the expression of these specific carbohydrate structures on any human cell. These vectors are variants of the pX330 construct described earlier ((1), http://crispr.genome-engineering.org/)
Transfection or electroporation of cells using these vectors results in efficient truncation of the following proteins ((2), Figure below):
About COSMC (C1GALT1C1): COSMC is a chaperone necessary for Core-1 Galactosyltransferase T-synthase (C1GalT1) activity. Its absence prevents the extension of core-2 and core-4 O-linked glycans (3) (Fig. 1a).
About MGAT-I: MGAT-I encodes for the enzyme GlcNAcT1. It is necessary for hybrid and complex N-glycan biosynthesis (4) (Fig. 1c). Absence of MGAT-I results in the expression of high-mannose structures.
About UGCG: UGCG exclusively adds the first glucose to ceramide substrates (5). Ablating this enzyme activity prevents glucosylceramide biosynthesis (Fig. 1b).
These plasmids were originally described in:
Stolfa, G., Mondal, N., Zhu, Y., Yu, X., Buffone, A. Jr. and Neelamegham, S. “Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and Glycosphingolipids to human leukocyte-endothelium adhesion. Scientific reports. 2016;6:30392.”
- Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 2013;339(6121):819-23.
- Stolfa G, Mondal N, Zhu Y, Yu X, Buffone A, Jr., Neelamegham S. Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and Glycosphingolipids to human leukocyte-endothelium adhesion. Scientific reports. 2016;6:30392.
- Ju T, Cummings RD. A unique molecular chaperone Cosmc required for activity of the mammalian core 1 beta 3-galactosyltransferase. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(26):16613-8.
- Chen W, Stanley P. Five Lec1 CHO cell mutants have distinct Mgat1 gene mutations that encode truncated N-acetylglucosaminyltransferase I. Glycobiology. 2003;13(1):43-50.
- Ichikawa S, Hirabayashi Y. Glucosylceramide synthase and glycosphingolipid synthesis. Trends in cell biology. 1998;8(5):198-202.