title: Modifying an Insect Cell N-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 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 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 N-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 N-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.
authors.affiliation: Department of Molecular Biology, University of Wyoming , Laramie, Wyoming 82071, United States.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Mabashi-Asazuma
authors.affiliation: Institute of Biological Chemistry, Academia Sinica 128 Nankang, Taipei 115, Taiwan.
authors.forename: Chu-Wei
authors.initials: CW
authors.lastname: Kuo
authors.affiliation: Institute of Biological Chemistry, Academia Sinica 128 Nankang, Taipei 115, Taiwan.
authors.forename: Kay-Hooi
authors.initials: KH
authors.lastname: Khoo
authors.affiliation: Department of Molecular Biology, University of Wyoming , Laramie, Wyoming 82071, United States.|GlycoBac, LLC, Laramie, Wyoming 82072, United States.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Jarvis
mesh_terms: D000118:Acetylglucosaminidase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D053843:DNA Mismatch Repair; D029721:Drosophila Proteins / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D008969:Molecular Sequence Data; D009154:Mutation; D011134:Polysaccharides / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics
chemical_list: D029721:Drosophila Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D000118:Acetylglucosaminidase; C509916:fdl protein, Drosophila