Journal
ACS CHEMICAL BIOLOGY
Volume 10, Issue 10, Pages 2199-2208Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acschembio.5b00340
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Funding
- National Institute of General Medical Sciences [R43GM102982]
- Taiwan National Core Facility Program for Biotechnology, NSC [NSC102-2319-B-001-003]
- NIH [2P40OD010949-10A1]
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Fused lobes (FDL) is an enzyme that simultaneously catalyzes a key trimming reaction and antagonizes elongation reactions in the insect N-glycan processing pathway. Accordingly, FDL function accounts, at least in part, for major differences in the N-glycosylation patterns of glycoproteins produced by insect and mammalian cells. In this study, we used the CRISPR-Cas9 system to edit the fdl gene in Drosophila melanogaster S-2 cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of insect-type, paucimannosidic products (Man(3)GlcNAc(2)), and led to the production of partially elongated, mammalian-type complex N-glycans (GlcNAc(2)Man(3)GlcNAc(2)) in S-2 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 N-glycan processing pathways. Finally, the new FDL-deficient S-2 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.
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