title: Inactivation of GDP-fucose transporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies.
abstract: Removal of core fucose from N-glycans attached to human IgG1 significantly enhances its affinity for the receptor FcγRIII and thereby dramatically improves its antibody-dependent cellular cytotoxicity activity. While previous works have shown that inactivation of fucosyltransferase 8 results in mutants capable of producing fucose-free antibodies, we report here the use of genome editing techniques, namely ZFNs, TALENs and the CRISPR-Cas9, to inactivate the GDP-fucose transporter (SLC35C1) in Chinese hamster ovary (CHO) cells. A FACS approach coupled with a fucose-specific lectin was developed to rapidly isolate SLC35C1-deficient cells. Mass spectrometry analysis showed that both EPO-Fc produced in mutants arising from CHO-K1 and anti-Her2 antibody produced in mutants arising from a pre-existing antibody-producing CHO-HER line lacked core fucose. Lack of functional SLC35C1 in these cells does not affect cell growth or antibody productivity. Our data demonstrate that inactivating Slc35c1 gene represents an alternative approach to generate CHO cells for production of fucose-free antibodies.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.|Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Kah Fai
authors.initials: KF
authors.lastname: Chan
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Wahyu
authors.initials: W
authors.lastname: Shahreel
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Corrine
authors.initials: C
authors.lastname: Wan
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Gavin
authors.initials: G
authors.lastname: Teo
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Noor
authors.initials: N
authors.lastname: Hayati
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Shi Jie
authors.initials: SJ
authors.lastname: Tay
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Wen Han
authors.initials: WH
authors.lastname: Tong
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.|Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Peiqing
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore. song_zhiwei@bti.a-star.edu.sg.|Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. song_zhiwei@bti.a-star.edu.sg.
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Song
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D016466:CHO Cells; D064113:CRISPR-Cas Systems; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D005643:Fucose / Q000737:chemistry*; D020868:Gene Silencing; D006801:Humans; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009154:Mutation; D017452:Receptors, IgG / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D000911:Antibodies, Monoclonal; D009004:Monosaccharide Transport Proteins; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D005643:Fucose
keywords: Chinese hamster ovary (CHO) cells; Fluorescence-activated cell sorting (FACS); Fucose-free antibodies; GDP-fucose transporter gene (Slc35c1); Genome editing technologies