Modifying an Insect Cell <i>N</i>‑Glycan Processing Pathway Using CRISPR-Cas Technology

Abstract

Fused lobes (FDL) is an enzyme that simultaneously catalyzes a key trimming reaction and antagonizes elongation reactions in the insect <i>N</i>-glycan processing pathway. Accordingly, FDL function accounts, at least in part, for major differences in the <i>N</i>-glycosylation patterns of glycoproteins produced by insect and mammalian cells. In this study, we used the CRISPR-Cas9 system to edit the <i>fdl</i> gene in Drosophila melanogaster S2 cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of insect-type, paucimannosidic products (Man₃GlcNAc₂), and led to the production of partially elongated, mammalian-type complex <i>N</i>-glycans (GlcNAc₂Man₃GlcNAc₂) in S2 cells. As CRISPR-Cas9 has not been widely used to analyze or modify protein glycosylation pathways or edit insect cell genes, these results underscore its broad utility as a tool for these purposes. Our results also confirm the key role of FDL at the major branch point distinguishing insect and mammalian <i>N</i>-glycan processing pathways. Finally, the new FDL-deficient S2 cell derivative produced in this study will enable future bottom-up glycoengineering efforts designed to isolate insect cell lines that can efficiently produce recombinant glycoproteins with chemically predefined oligosaccharide side-chain structures