Characterization of Glycogene Knockout

Glycans are formed on the surface of human/mammalian cells by the combined action of various families of "glycogen" and corresponding "glycol-enzyme". The final structure and function of the carbohydrates formed in the cell, depend on the underlying cellular glycogenetic composition and mechanism. In addition, the glycogenes involved in the formation of specific carbohydrate structures may vary across cell types. Complete knockout is achieved by inducing DNA double-stranding to produce double-strand breaks. It utilizes the organism's non-homologous end-joining-mediated deletion of large segments of the gene reading frame to induce loss of function of the gene. Combining glycosylation pathways with gene expression data that glycans can be regulated by gene expression and substrate accumulation levels. The preparation of a large number of knockout cell models and animal models for probing the function of genes, and the course of human disease has greatly contributed to the development of current biotechnology.

Fig.1 Generation of Lafora disease model mice with tamoxifen-inducible Gys1 knockout. (Nitschke, et al., 2021)

Glycogene Knockout Service at CD BioGlyco

With the help of various gene editing tools and different novel knockout technologies, CD BioGlyco provides fast, accurate, and efficient glycogene knockout services to clients all over the world. It helps our clients to better understand the function of glycosylation modification, the process of signal transduction. Discover new glycan biomarkers and therapeutic targets by identifying the differences in glycan structure between normal expression and knockout expression.

• Glycogene knockout methods
  
  • Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system: contains Cas9 nuclease and single guide RNA (sgRNA) targeting sequences to enable mutation and knockout of target genes by pinpointing the DNA sequence of the target gene and introducing double-strand breaks.
  • Transcription activation-like effector nucleases (TALENs) vectors: contain specific nuclease and nuclease binding domains for knockdown and editing by cleavage of DNA sequences of target genes.
  • Zinc finger nucleases (ZFNs) vectors: contain zinc finger structural domains and nucleases with specificity for knockout and editing by cleaving DNA sequences of target genes.

We improve knockout efficiency and reduce off-target effects by double incision technology. Our clients only need to provide the glycogen knockout site, and we provide you with a one-stop service process. The service from primer design/model preparation/amplification sequencing or western blot analysis, etc., to genotype detection and glycosylation modification.

Fig.2 The flowchart of glycogene knockout. (CD BioGlyco)

Applications

• Animal husbandry: glycogene knockout plays a key role in livestock breeding, it improves fertility and disease resistance.
• Multilocus glycogene knockout in rabbits lays the foundation for disease cure imitating the human genome.
• Visualizing and estimating glycan structures based on gene knockouts.
• Estimation of glycan changes between normal and diseased tissues based on knockout of specific glycogen genes.

Advantages

• The knockout still edits the epigenetic modifications of genes, such as methylation.
• Equipped with the Glycogenomics Platform, a professional scientific team and many years of proven experience at CD BioGlyco provides high-efficiency knockout of the target gene service.
• We utilize a comprehensive network of gRNA design tools to improve accuracy using a dual gRNA system with over 95% efficiency.
• Adequate genomic and proteomic validation using highly specific recombinant monoclonal antibodies confirms glycogene knockout.

CD BioGlyco has experienced gene editing technicians who follow you all the way. We are capable of providing efficient and reliable services to accelerate our clients' scientific research by shortening the project time. If you are interested in our Glycogene Editing Technology or Glycogene Engineering Service, please feel free to contact us, and honored to discuss with you further.

References:

1. Kelkar, A.; et al. Forward genetic screens of human glycosylation pathways using the glycoGene CRISPR library. Curr Protoc. 2022, 2(4): e402.
2. Huang, Y.F.; et al. Global mapping of glycosylation pathways in human-derived cells. Dev Cell. 2021, 56(8): 1195-1209.
3. Nitschke, S.; et al. An inducible glycogen synthase-1 knockout halts but does not reverse Lafora disease progression in mice. J Biol Chem. 2021, 296: 100150.