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Genetic glycoengineering in mammalian cells

  • Yoshiki Narimatsu
    Correspondence
    For correspondence: Yoshiki Narimatsu; Christian Büll; Henrik Clausen
    Affiliations
    Department of Cellular and Molecular Medicine, Faculty of Health Sciences, Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen, Denmark

    GlycoDisplay ApS, Copenhagen, Denmark
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  • Christian Büll
    Correspondence
    For correspondence: Yoshiki Narimatsu; Christian Büll; Henrik Clausen
    Affiliations
    Department of Cellular and Molecular Medicine, Faculty of Health Sciences, Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen, Denmark
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  • Yen-Hsi Chen
    Affiliations
    GlycoDisplay ApS, Copenhagen, Denmark
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  • Hans H. Wandall
    Affiliations
    Department of Cellular and Molecular Medicine, Faculty of Health Sciences, Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen, Denmark
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  • Zhang Yang
    Affiliations
    Department of Cellular and Molecular Medicine, Faculty of Health Sciences, Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen, Denmark

    GlycoDisplay ApS, Copenhagen, Denmark
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  • Henrik Clausen
    Correspondence
    For correspondence: Yoshiki Narimatsu; Christian Büll; Henrik Clausen
    Affiliations
    Department of Cellular and Molecular Medicine, Faculty of Health Sciences, Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen, Denmark
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Open AccessPublished:February 19, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100448
      Advances in nuclease-based gene-editing technologies have enabled precise, stable, and systematic genetic engineering of glycosylation capacities in mammalian cells, opening up a plethora of opportunities for studying the glycome and exploiting glycans in biomedicine. Glycoengineering using chemical, enzymatic, and genetic approaches has a long history, and precise gene editing provides a nearly unlimited playground for stable engineering of glycosylation in mammalian cells to explore and dissect the glycome and its many biological functions. Genetic engineering of glycosylation in cells also brings studies of the glycome to the single cell level and opens up wider use and integration of data in traditional omics workflows in cell biology. The last few years have seen new applications of glycoengineering in mammalian cells with perspectives for wider use in basic and applied glycosciences, and these have already led to discoveries of functions of glycans and improved designs of glycoprotein therapeutics. Here, we review the current state of the art of genetic glycoengineering in mammalian cells and highlight emerging opportunities.

      Keywords

      Abbreviations:

      ADCC (antibody-dependent cell cytotoxicity), CHO (Chinese hamster ovary), CRISPR (clustered regularly interspaced short palindromic repeat), EPO (erythropoietin), ES (embryonic stem), GBP (glycan-binding protein), GT (glycosyltransferase), HA (hemagglutinin), HDR (homology-directed repair), KI (knock-in), KO (knockout), MMEJ (microhomology-mediated end joining), NHEJ (nonhomologous end joining), PAM (proto-spacer adjacent motif), TALEN (transcription activator-like effector nuclease), TR (tandem repeat), ZFN (zinc-finger nuclease)
      Engineering glycosylation in cells and organisms has a long history (
      • Stanley P.
      What have we learned from glycosyltransferase knockouts in mice?.
      ,
      • Lowe J.B.
      • Marth J.D.
      A genetic approach to mammalian glycan function.
      ,
      • Orr S.L.
      • Le D.
      • Long J.M.
      • Sobieszczuk P.
      • Ma B.
      • Tian H.
      • Fang X.
      • Paulson J.C.
      • Marth J.D.
      • Varki N.
      A phenotype survey of 36 mutant mouse strains with gene-targeted defects in glycosyltransferases or glycan-binding proteins.
      ), and yet rational genetic engineering of the glycosylation capacities in mammalian cells may only be off to a start (
      • Steentoft C.
      • Bennett E.P.
      • Schjoldager K.T.
      • Vakhrushev S.Y.
      • Wandall H.H.
      • Clausen H.
      Precision genome editing: A small revolution for glycobiology.
      ) (Fig. 1). The glycosylation machinery in cells involves a large and complex metabolic network of enzymes and accessory proteins that orchestrate the synthesis of different types of glycans found on glycolipids, glycoproteins, proteoglycans, and as oligosaccharides. Over 200 distinct glycosyltransferase (GT) genes contribute to glycosylation in mammalian cells, and at least 173 of these GTs function in at least 16 distinct glycosylation pathways to assemble the great diversity of glycan structures found on glycoproteins, glycolipids, and proteoglycans (
      • Schjoldager K.T.
      • Narimatsu Y.
      • Joshi H.J.
      • Clausen H.
      Global view of human protein glycosylation pathways and functions.
      ). Rational engineering of glycosylation in mammalian cells can now be performed with a high degree of confidence in predicted outcomes, and genetic engineering is one approach to studying the biology of glycans that takes origin in a single cell. However, challenges still exist with isoenzymes for which unique functions are poorly characterized and partial overlaps in properties need to be considered.
      Figure thumbnail gr1
      Figure 1Overview of glycoengineering strategies. Basic principles for approaches available to modulate the cellular glycosylation processes and the glycome are illustrated. Extracellularly, glycans may be modulated by more or less selective endo-/exo-glycosidases (sialidases, galactosidases, PNGase, etc.) (
      • Henrissat B.
      • Surolia A.
      • Stanley P.
      A genomic view of glycobiology.
      ), and chemoenzymatic labeling methods utilizing, e.g., glycosyltransferases (GTs) may be applied to install natural or unnatural substrates on cell surface glycans (
      • Griffin M.E.
      • Hsieh-Wilson L.C.
      Glycan engineering for cell and developmental biology.
      ,
      • Mbua N.E.
      • Li X.
      • Flanagan-Steet H.R.
      • Meng L.
      • Aoki K.
      • Moremen K.W.
      • Wolfert M.A.
      • Steet R.
      • Boons G.J.
      Selective exo-enzymatic labeling of N-glycans on the surface of living cells by recombinant ST6Gal I.
      ). Use of cytotoxic lectins often in combination with mutagenesis may enable selection of mutant cells with loss/gain of distinct glycosylation features (
      • Patnaik S.K.
      • Stanley P.
      Lectin-resistant CHO glycosylation mutants.
      ,
      • Esko J.D.
      • Stanley P.
      Glycosylation mutants of cultured cells.
      ). A growing number of unnatural sugar mimetics can be applied for metabolic engineering (
      • Griffin M.E.
      • Hsieh-Wilson L.C.
      Glycan engineering for cell and developmental biology.
      ,
      • Moons S.J.
      • Adema G.J.
      • Derks M.T.
      • Boltje T.J.
      • Bull C.
      Sialic acid glycoengineering using N-acetylmannosamine and sialic acid analogs.
      ), including glycosylation inhibitors (i.e., fluorinated sugar analogues) (
      • Rillahan C.D.
      • Antonopoulos A.
      • Lefort C.T.
      • Sonon R.
      • Azadi P.
      • Ley K.
      • Dell A.
      • Haslam S.M.
      • Paulson J.C.
      Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome.
      ,
      • Esko J.D.
      • Bertozzi C.
      • Schnaar R.L.
      Chemical tools for inhibiting glycosylation.
      ) or functionalized sugars (i.e., azido, Az, sugars) that enable conjugation chemistries for use in glycan imaging or reprogramming of their interactions (
      • Hudak J.E.
      • Bertozzi C.R.
      Glycotherapy: New advances inspire a reemergence of glycans in medicine.
      ,
      • Prescher J.A.
      • Bertozzi C.R.
      Chemical technologies for probing glycans.
      ). Genetic engineering of glycosylation may be performed by overexpression (OE) of GTs using cDNA plasmid transfection and/or siRNA for silencing of endogenous GTs (
      • Griffin M.E.
      • Hsieh-Wilson L.C.
      Glycan engineering for cell and developmental biology.
      ). More extensive and stable glycoengineering takes advantage of precise gene engineering for combinatorial KO/KI/Act (activation) of GTs, and this strategy is the main focus of this review. Genome-wide KO/Act screens (GWS) may be used for discovery and dissection of GTs and other genes affecting glycosylation (), and endogenous GTs may be mutated, e.g., to mimic disease mutations or enable use of unique substrates, or tagged, e.g., by insertion of antibody tags or fluorescent proteins (
      • Cioce A.
      • Malaker S.A.
      • Schumann B.
      Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools.
      ).
      Genetic engineering of glycosylation in mammalian cells has contributed tremendously to the current view of the genetic and biosynthetic regulation of the cellular glycome and highlighted important functions of glycans in development, health, and disease (
      • Stanley P.
      What have we learned from glycosyltransferase knockouts in mice?.
      ,
      • Lowe J.B.
      • Marth J.D.
      A genetic approach to mammalian glycan function.
      ,
      • Orr S.L.
      • Le D.
      • Long J.M.
      • Sobieszczuk P.
      • Ma B.
      • Tian H.
      • Fang X.
      • Paulson J.C.
      • Marth J.D.
      • Varki N.
      A phenotype survey of 36 mutant mouse strains with gene-targeted defects in glycosyltransferases or glycan-binding proteins.
      ,
      • Schjoldager K.T.
      • Narimatsu Y.
      • Joshi H.J.
      • Clausen H.
      Global view of human protein glycosylation pathways and functions.
      ,
      • Griffin M.E.
      • Hsieh-Wilson L.C.
      Glycan engineering for cell and developmental biology.
      ). Early on mammalian mutant cell lines with loss of GT genes or other genes affecting glycosylation were generated by random mutagenesis and selection for lectin resistance (
      • Gottlieb C.
      • Skinner A.M.
      • Kornfeld S.
      Isolation of a clone of Chinese hamster ovary cells deficient in plant lectin-binding sites.
      ,
      • Gottlieb C.
      • Kornfeld S.
      Isolation and characterization of two mouse L cell lines resistant to the toxic lectin ricin.
      ,
      • Stanley P.
      Lectin-resistant CHO cells: Selection of new mutant phenotypes.
      ,
      • Patnaik S.K.
      • Stanley P.
      Lectin-resistant CHO glycosylation mutants.
      ), and later targeted knockout (KO) of GT genes in animals by homologous gene recombination strategies in embryonic stem (ES) cells opened up rational design of glycoengineered models (
      • Stanley P.
      What have we learned from glycosyltransferase knockouts in mice?.
      ,
      • Lowe J.B.
      • Marth J.D.
      A genetic approach to mammalian glycan function.
      ,
      • Orr S.L.
      • Le D.
      • Long J.M.
      • Sobieszczuk P.
      • Ma B.
      • Tian H.
      • Fang X.
      • Paulson J.C.
      • Marth J.D.
      • Varki N.
      A phenotype survey of 36 mutant mouse strains with gene-targeted defects in glycosyltransferases or glycan-binding proteins.
      ). These models informed about GT genes that are essential for early development of the embryo and genes with important functions for normal health and diseases, but also of many GT genes that at first pass at least appeared to be dispensable for normal development and health. These studies also highlighted that some steps in the glycosylation pathways in mammalian cells are regulated by a single GT with loss of function of such genes leading to global changes in the glycan structures produced. However, these studies also revealed glycosylation steps that are regulated by multiple isoenzymes with at least partly redundant functions, where loss of function of a single isoenzyme gene may lead to no or only subtle effects on the glycans (
      • Ohtsubo K.
      • Marth J.D.
      Glycosylation in cellular mechanisms of health and disease.
      ,
      • Marth J.D.
      Complexity in O-linked oligosaccharide biosynthesis engendered by multiple polypeptide N-acetylgalactosaminyltransferases.
      ,
      • Yang W.H.
      • Nussbaum C.
      • Grewal P.K.
      • Marth J.D.
      • Sperandio M.
      Coordinated roles of ST3Gal-VI and ST3Gal-IV sialyltransferases in the synthesis of selectin ligands.
      ,
      • Takamatsu S.
      • Antonopoulos A.
      • Ohtsubo K.
      • Ditto D.
      • Chiba Y.
      • Le D.T.
      • Morris H.R.
      • Haslam S.M.
      • Dell A.
      • Marth J.D.
      • Taniguchi N.
      Physiological and glycomic characterization of N-acetylglucosaminyltransferase-IVa and -IVb double deficient mice.
      ,
      • Stone E.L.
      • Ismail M.N.
      • Lee S.H.
      • Luu Y.
      • Ramirez K.
      • Haslam S.M.
      • Ho S.B.
      • Dell A.
      • Fukuda M.
      • Marth J.D.
      Glycosyltransferase function in core 2-type protein O glycosylation.
      ).
      A major obstacle for the wider use of genetic engineering of glycosylation in mammalian cells has until recently been the lack of simple and efficient means to design and conduct rational and combinatorial engineering of genes. In lieu of this, considerable efforts have been devoted to engineering glycosylation in other eukaryotic cells such as yeast, plants, and insects where tools for genetic engineering were more readily available (
      • Van Landuyt L.
      • Lonigro C.
      • Meuris L.
      • Callewaert N.
      Customized protein glycosylation to improve biopharmaceutical function and targeting.
      ,
      • Montero-Morales L.
      • Steinkellner H.
      Advanced plant-based glycan engineering.
      ,
      • Naegeli A.
      • Aebi M.
      Current approaches to engineering N-linked protein glycosylation in bacteria.
      ,
      • Laukens B.
      • De Visscher C.
      • Callewaert N.
      Engineering yeast for producing human glycoproteins: Where are we now?.
      ). Engineering of glycosylation has also been sought by a wide range of nongenetic strategies including chemical and enzymatic approaches (Fig. 1) (
      • Griffin M.E.
      • Hsieh-Wilson L.C.
      Glycan engineering for cell and developmental biology.
      ,
      • Hudak J.E.
      • Bertozzi C.R.
      Glycotherapy: New advances inspire a reemergence of glycans in medicine.
      ,
      • Kightlinger W.
      • Warfel K.F.
      • DeLisa M.P.
      • Jewett M.C.
      Synthetic glycobiology: Parts, systems, and applications.
      ,
      • Agatemor C.
      • Buettner M.J.
      • Ariss R.
      • Muthiah K.
      • Saeui C.T.
      • Yarema K.J.
      Exploiting metabolic glycoengineering to advance healthcare.
      ,
      • Moons S.J.
      • Adema G.J.
      • Derks M.T.
      • Boltje T.J.
      • Bull C.
      Sialic acid glycoengineering using N-acetylmannosamine and sialic acid analogs.
      ). The primary objectives for genetic engineering of glycosylation were devoted to glycoprotein therapeutics, and the first targeted KO engineering in a mammalian cell involved a tour-de-force effort performed using two rounds of targeted homologous recombination in Chinese hamster ovary (CHO) cells to knockout the two copies of the α6-fucosyltransferase (fut8) gene. FUT8 directs core fucosylation of N-glycans and the CHO fut8 KO cells enabled production of afucosylated IgG with greatly improved antibody-dependent cell cytotoxicity (ADCC) (
      • Yamane-Ohnuki N.
      • Kinoshita S.
      • Inoue-Urakubo M.
      • Kusunoki M.
      • Iida S.
      • Nakano R.
      • Wakitani M.
      • Niwa R.
      • Sakurada M.
      • Uchida K.
      • Shitara K.
      • Satoh M.
      Establishment of FUT8 knockout Chinese hamster ovary cells: An ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.
      ,
      • Malphettes L.
      • Freyvert Y.
      • Chang J.
      • Liu P.Q.
      • Chan E.
      • Miller J.C.
      • Zhou Z.
      • Nguyen T.
      • Tsai C.
      • Snowden A.W.
      • Collingwood T.N.
      • Gregory P.D.
      • Cost G.J.
      Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies.
      ). However, the huge efforts required with this strategy limit broader use for engineering mammalian cell lines.
      A new era for glycoengineering emerged with the introduction of the facile nuclease-based gene-editing strategies at the turn of the millennium (
      • Steentoft C.
      • Bennett E.P.
      • Schjoldager K.T.
      • Vakhrushev S.Y.
      • Wandall H.H.
      • Clausen H.
      Precision genome editing: A small revolution for glycobiology.
      ,
      • Doudna J.A.
      The promise and challenge of therapeutic genome editing.
      ). The zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats with the CRISPR-associated protein 9 (CRISPR/Cas9) tools now allow for rational designed gene engineering events in mammalian cells with high precision, speed, and low cost. Engineering events in cells can be stacked with multiple gene KOs in combination with knock-ins (KIs) to achieve almost any desirable design of the cellular glycosylation capacities. These new opportunities are beginning to thrive, and engineering of glycosylation in cell lines is no longer only focused on meeting interests in improved glycoprotein therapeutics. Already a remarkable diversity in the use of glycoengineering has emerged. Here, we focus on these new advances and discuss the use of strategies to comprehensively engineer and dissect glycosylation in mammalian cell lines, which we define as rational genetic engineering of glycosylation. A number of excellent reviews have already summarized and discussed progress with engineering glycosylation in different species including yeast, insects, and plants and the special opportunities these provide for production of glycoprotein therapeutics (
      • Griffin M.E.
      • Hsieh-Wilson L.C.
      Glycan engineering for cell and developmental biology.
      ,
      • Van Landuyt L.
      • Lonigro C.
      • Meuris L.
      • Callewaert N.
      Customized protein glycosylation to improve biopharmaceutical function and targeting.
      ,
      • Montero-Morales L.
      • Steinkellner H.
      Advanced plant-based glycan engineering.
      ,
      • Naegeli A.
      • Aebi M.
      Current approaches to engineering N-linked protein glycosylation in bacteria.
      ,
      • Laukens B.
      • De Visscher C.
      • Callewaert N.
      Engineering yeast for producing human glycoproteins: Where are we now?.
      ,
      • Hudak J.E.
      • Bertozzi C.R.
      Glycotherapy: New advances inspire a reemergence of glycans in medicine.
      ,
      • Kightlinger W.
      • Warfel K.F.
      • DeLisa M.P.
      • Jewett M.C.
      Synthetic glycobiology: Parts, systems, and applications.
      ,
      • Agatemor C.
      • Buettner M.J.
      • Ariss R.
      • Muthiah K.
      • Saeui C.T.
      • Yarema K.J.
      Exploiting metabolic glycoengineering to advance healthcare.
      ,
      • Wang L.X.
      • Amin M.N.
      Chemical and chemoenzymatic synthesis of glycoproteins for deciphering functions.
      ,
      • Mizukami A.
      • Caron A.L.
      • Picanco-Castro V.
      • Swiech K.
      Platforms for recombinant therapeutic glycoprotein production.
      ,
      • Dicker M.
      • Strasser R.
      Using glyco-engineering to produce therapeutic proteins.
      ).

      Glycosylation processes in cells

      The glycosylation machinery of a given cell determines the ensemble of glycan structures and types of glycoconjugates that constitute the glycome of that same cell (
      • Cummings R.D.
      The repertoire of glycan determinants in the human glycome.
      ). The glycosylation capacities of different cell types vary primarily through changes in expression of the GTs that directs different steps in glycosylation pathways (Fig. 2A); however, a number of other factors affect the glycosylation outcome (
      • Schjoldager K.T.
      • Narimatsu Y.
      • Joshi H.J.
      • Clausen H.
      Global view of human protein glycosylation pathways and functions.
      ,
      • Moremen K.W.
      • Tiemeyer M.
      • Nairn A.V.
      Vertebrate protein glycosylation: Diversity, synthesis and function.
      ). GTs have different donor sugar and acceptor substrate specificities often with considerable overlaps (
      • Moremen K.W.
      • Haltiwanger R.S.
      Emerging structural insights into glycosyltransferase-mediated synthesis of glycans.
      ), and glycosylation reactions are primarily guided by the kinetic properties of the expressed ensemble of enzymes (
      • Lowe J.B.
      • Marth J.D.
      A genetic approach to mammalian glycan function.
      ,
      • Narimatsu Y.
      • Joshi H.J.
      • Nason R.
      • Van Coillie J.
      • Karlsson R.
      • Sun L.
      • Ye Z.
      • Chen Y.H.
      • Schjoldager K.T.
      • Steentoft C.
      • Furukawa S.
      • Bensing B.A.
      • Sullam P.M.
      • Thompson A.J.
      • Paulson J.C.
      • et al.
      An atlas of human glycosylation pathways enables display of the human glycome by gene engineered cells.
      ,
      • Nairn A.V.
      • York W.S.
      • Harris K.
      • Hall E.M.
      • Pierce J.M.
      • Moremen K.W.
      Regulation of glycan structures in animal tissues: Transcript profiling of glycan-related genes.
      ). Glycosylation is therefore akin to a large integrated metabolic network involving a myriad of enzyme reactions, where a number of “rules” can be applied to predict the outcomes in terms of glycans being produced. While many of these rules for enzyme reactions can be applied with a high degree of fidelity, others involve less predictable outcomes. Thus, rational engineering of glycosylation in cells requires information on glycosylation processes and often experimentation and validation of outcomes.
      Figure thumbnail gr2
      Figure 2Overview of principles and strategies for stable genetic engineering of cellular glycosylation capacities. A, overview of the 16 human glycosylation pathways with predicted assignments of 173 glycosyltransferase genes to the major biosynthetic steps using the rainbow display organization (
      • Schjoldager K.T.
      • Narimatsu Y.
      • Joshi H.J.
      • Clausen H.
      Global view of human protein glycosylation pathways and functions.
      ,
      • Bull C.
      • Joshi H.J.
      • Clausen H.
      • Narimatsu Y.
      Cell-based glycan arrays-a practical guide to dissect the human glycome.
      ). The rainbow depiction of glycosylation pathways illustrates the major biosynthetic steps organized into pathway-specific steps (right part with even colored according to the initial monosaccharide except for glycolipids) and pathway nonspecific steps (left part with toned colors) with predicted GT genes assigned. Note that this is a simplified scheme of pathways and GT genes are assigned only to the primary predicted functions. Genetic engineering of glycosylation requires considering the properties of individual enzymes and their potential effects on the cellular glycosylation pathways. Loss or gain of a GT may have highly specific effects or wider effects on multiple glycosylation pathways. Glycosylation steps covered exclusively by one unique enzyme (nonredundant steps), e.g., core α6-fucosylation of N-glycans by FUT8, yield highly specific and predictable outcomes with loss/gain engineering. Steps covered by multiple isoenzymes with overlapping functions (redundant steps) may or may not yield easily predictable outcomes, and the outcome may vary in cells dependent on the expression of such isoenzymes. Most steps in elongation and branching and capping are covered by partial redundancies by multiple isoenzymes. For example, sialylation by any of the four sialyltransferase subfamilies is covered by partial redundancies, and, e.g., combinatorial KO of three genes is required to selectively eliminate α3-sialylation on N-glycans (KO of ST3GAL3/4/6), while KO of two genes is required to selectively eliminate α3-sialylation of core1 O-glycans (KO ST3GAL1/2). Glycan symbols are displayed in the Symbol Nomenclature for Glycans (SNFG) format (
      • Varki A.
      • Cummings R.D.
      • Aebi M.
      • Packer N.H.
      • Seeberger P.H.
      • Esko J.D.
      • Stanley P.
      • Hart G.
      • Darvill A.
      • Kinoshita T.
      • Prestegard J.J.
      • Schnaar R.L.
      • Freeze H.H.
      • Marth J.D.
      • Bertozzi C.R.
      • et al.
      Symbol nomenclature for graphical representations of glycans.
      ). B, graphic depiction of current nuclease-based gene-editing tools for knockout (KO) and knock-in (KI) of genes, and emerging CRISPR-based technologies for regulating and activating gene expression.
      Figure 2A organizes glycosylation into 16 distinct pathways and major biosynthetic steps with assignment of predicted roles of 173 human GTs (note that we refer to GTs by their gene names only for simplicity; capital letters refer to human GTs, and italics refer to genes) (
      • Schjoldager K.T.
      • Narimatsu Y.
      • Joshi H.J.
      • Clausen H.
      Global view of human protein glycosylation pathways and functions.
      ,
      • Narimatsu Y.
      • Joshi H.J.
      • Nason R.
      • Van Coillie J.
      • Karlsson R.
      • Sun L.
      • Ye Z.
      • Chen Y.H.
      • Schjoldager K.T.
      • Steentoft C.
      • Furukawa S.
      • Bensing B.A.
      • Sullam P.M.
      • Thompson A.J.
      • Paulson J.C.
      • et al.
      An atlas of human glycosylation pathways enables display of the human glycome by gene engineered cells.
      ,
      • Joshi H.J.
      • Narimatsu Y.
      • Schjoldager K.T.
      • Tytgat H.L.P.
      • Aebi M.
      • Clausen H.
      • Halim A.
      SnapShot: O-glycosylation pathways across kingdoms.
      ). The map provides a blueprint for rational engineering of glycosylation in mammalian cells and highlight: i) GTs that are unique and serve glycosylation pathway-specific roles (essentially all steps in initiation of glycosylation on lipids and proteins and most early steps in core extension) and GTs that serve pathway nonspecific roles (assembly of elongated and branched glycans and the final capping); and ii) glycosylation steps that are covered by single nonredundant GTs and those that are covered by partial redundancies from GT isoenzymes. Moreover, the map serves to translate cellular expression data for GTs from, e.g., single-cell transcriptomics or proteomics into predicted glycosylation capacities and glycome outcomes, as recently demonstrated (
      • Narimatsu Y.
      • Joshi H.J.
      • Nason R.
      • Van Coillie J.
      • Karlsson R.
      • Sun L.
      • Ye Z.
      • Chen Y.H.
      • Schjoldager K.T.
      • Steentoft C.
      • Furukawa S.
      • Bensing B.A.
      • Sullam P.M.
      • Thompson A.J.
      • Paulson J.C.
      • et al.
      An atlas of human glycosylation pathways enables display of the human glycome by gene engineered cells.
      ). The glycosylation capacities of immortalized mammalian cell lines such as the CHO and HEK293 cells widely used for recombinant expression of glycoproteins can be extensively manipulated by multiple KO/KI events without major effects on viability in simple cultures and performance in production and secretion (
      • Narimatsu Y.
      • Joshi H.J.
      • Nason R.
      • Van Coillie J.
      • Karlsson R.
      • Sun L.
      • Ye Z.
      • Chen Y.H.
      • Schjoldager K.T.
      • Steentoft C.
      • Furukawa S.
      • Bensing B.A.
      • Sullam P.M.
      • Thompson A.J.
      • Paulson J.C.
      • et al.
      An atlas of human glycosylation pathways enables display of the human glycome by gene engineered cells.
      ). The only essential steps for cell viability may be complete loss of the function of the oligosaccharyltransferase (OST) complex and initiation of N-glycosylation and complete loss of the cytosolic O-GlcNAcylation orchestrated by the protein O-GlcNAc transferase (OGT) (
      • O'Donnell N.
      • Zachara N.E.
      • Hart G.W.
      • Marth J.D.
      Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability.
      ,
      • Shafi R.
      • Iyer S.P.
      • Ellies L.G.
      • O'Donnell N.
      • Marek K.W.
      • Chui D.
      • Hart G.W.
      • Marth J.D.
      The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny.
      ).
      A major incentive for using genetic engineering of glycosylation in studying the biology of the glycome is that this is one strategy that allows probing the glycome at the single cell level. Engineering glycosylation capacities may be used to explore biological functions of glycosylation in a cell by dissection of the glycosylation genes and enzymes required for the particular function. Knowledge of the glycosylation pathways in the cell may then be used to predict the structural features of the glycome that underlies the function. Emerging high-quality single-cell transcriptomics and proteomics data can also be used to gauge the expression of glycosylation enzymes and with some confidence used to predict the cellular glycosylation capacities and glycome outcome (
      • Schjoldager K.T.
      • Narimatsu Y.
      • Joshi H.J.
      • Clausen H.
      Global view of human protein glycosylation pathways and functions.
      ). This information may be incorporated in engineering strategies to simplify and focus the design of genetic engineering on relevant active glycosylation pathways. Currently, analytic glycome and glycomics technologies are not at this level of sensitivity, and structural analytic data sets are generally sums of glycome contributions from multiple cells often with heterogeneous origins (
      • Cummings R.D.
      • Pierce J.M.
      The challenge and promise of glycomics.
      ,
      • Rudd P.
      • Karlsson N.G.
      • Khoo K.H.
      • Packer N.H.
      Glycomics and glycoproteomics.
      ). The glycome of any cell at a given state and timepoint is predicted to be less heterogeneous than current analytics suggest, and distinct glycosylation features may be highly regulated (
      • Nairn A.V.
      • York W.S.
      • Harris K.
      • Hall E.M.
      • Pierce J.M.
      • Moremen K.W.
      Regulation of glycan structures in animal tissues: Transcript profiling of glycan-related genes.
      ,
      • Park D.D.
      • Xu G.
      • Wong M.
      • Phoomak C.
      • Liu M.
      • Haigh N.E.
      • Wongkham S.
      • Yang P.
      • Maverakis E.
      • Lebrilla C.B.
      Membrane glycomics reveal heterogeneity and quantitative distribution of cell surface sialylation.
      ,
      • Fujitani N.
      • Furukawa J.
      • Araki K.
      • Fujioka T.
      • Takegawa Y.
      • Piao J.
      • Nishioka T.
      • Tamura T.
      • Nikaido T.
      • Ito M.
      • Nakamura Y.
      • Shinohara Y.
      Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers.
      ,
      • An H.J.
      • Gip P.
      • Kim J.
      • Wu S.
      • Park K.W.
      • McVaugh C.T.
      • Schaffer D.V.
      • Bertozzi C.R.
      • Lebrilla C.B.
      Extensive determination of glycan heterogeneity reveals an unusual abundance of high mannose glycans in enriched plasma membranes of human embryonic stem cells.
      ). Decades of studies with glycan-binding proteins (GBPs) including lectins and antibodies have shown that the expression and distribution of a limited number of glycan epitopes or structural glycan features change characteristically during cellular maturation and differentiation, tissue formation, and in diseases (
      • Riley N.M.
      • Hebert A.S.
      • Westphall M.S.
      • Coon J.J.
      Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis.
      ,
      • Freeze H.H.
      • Baum L.
      • Varki A.
      Glycans in systemic physiology.
      ,
      • Nairn A.V.
      • Aoki K.
      • dela Rosa M.
      • Porterfield M.
      • Lim J.M.
      • Kulik M.
      • Pierce J.M.
      • Wells L.
      • Dalton S.
      • Tiemeyer M.
      • Moremen K.W.
      Regulation of glycan structures in murine embryonic stem cells: Combined transcript profiling of glycan-related genes and glycan structural analysis.
      ,
      • Dabelsteen E.
      • Vedtofte P.
      • Hakomori S.I.
      • Young W.W.
      Carbohydrate chains specific for blood group antigens in differentiation of human oral epithelium.
      ,
      • Bull C.
      • Stoel M.A.
      • den Brok M.H.
      • Adema G.J.
      Sialic acids sweeten a tumor's life.
      ). Despite progress though, we are still limited in our abilities to decipher the molecular basis of specific roles of glycosylation in biological functions, and genetic glycoengineering in mammalian cells is one promising strategy for discovery and dissection.

      The tools—precise genome-editing techniques

      As pointed out above, genetic engineering of glycosylation in mammalian cells has been performed for decades. Considerable efforts have been devoted to overexpression and/or silencing of GTs (and other metabolic enzymes, transporters, hydrolases) in mammalian cells (
      • Mottram L.
      • Liu J.
      • Chavan S.
      • Tobias J.
      • Svennerholm A.M.
      • Holgersson J.
      Glyco-engineered cell line and computational docking studies reveals enterotoxigenic Escherichia coli CFA/I fimbriae bind to Lewis a glycans.
      ,
      • Lindberg L.
      • Liu J.
      • Holgersson J.
      Engineering of therapeutic and diagnostic O-glycans on recombinant mucin-type immunoglobulin fusion proteins expressed in CHO cells.
      ,
      • Li F.
      • Wilkins P.P.
      • Crawley S.
      • Weinstein J.
      • Cummings R.D.
      • McEver R.P.
      Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin.
      ,
      • Lee E.U.
      • Roth J.
      • Paulson J.C.
      Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of beta-galactoside alpha 2,6-sialyltransferase.
      ,
      • Shi W.X.
      • Chammas R.
      • Varki A.
      Linkage-specific action of endogenous sialic acid O-acetyltransferase in Chinese hamster ovary cells.
      ,
      • Mondal N.
      • Dykstra B.
      • Lee J.
      • Ashline D.J.
      • Reinhold V.N.
      • Rossi D.J.
      • Sackstein R.
      Distinct human alpha(1,3)-fucosyltransferases drive Lewis-X/sialyl Lewis-X assembly in human cells.
      ). However, overexpression of GTs may lead to markedly perturbed glycosylation (
      • Sewell R.
      • Backstrom M.
      • Dalziel M.
      • Gschmeissner S.
      • Karlsson H.
      • Noll T.
      • Gatgens J.
      • Clausen H.
      • Hansson G.C.
      • Burchell J.
      • Taylor-Papadimitriou J.
      The ST6GalNAc-I sialyltransferase localizes throughout the Golgi and is responsible for the synthesis of the tumor-associated sialyl-Tn O-glycan in human breast cancer.
      ), and gene silencing using RNA interference strategies often results in limited and variable reduction of enzyme levels with uncertain effects on glycosylation outcomes (
      • Steentoft C.
      • Bennett E.P.
      • Schjoldager K.T.
      • Vakhrushev S.Y.
      • Wandall H.H.
      • Clausen H.
      Precision genome editing: A small revolution for glycobiology.
      ,
      • Jackson A.L.
      • Linsley P.S.
      Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application.
      ,
      • Aagaard L.
      • Rossi J.J.
      RNAi therapeutics: Principles, prospects and challenges.
      ). Today, glycoengineering with precision genome-editing techniques overcomes these issues and enables stable genetic changes to the cellular glycosylation machinery. The rapidly expanding toolbox for precise genome editing offers wide opportunities for targeted KO, KI, or modulation of the expression of glycogenes to reprogram glycosylation in mammalian cells. In the following, we briefly review these tools and refer to original studies for details (
      • Jinek M.
      • Chylinski K.
      • Fonfara I.
      • Hauer M.
      • Doudna J.A.
      • Charpentier E.
      A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
      ,
      • Cong L.
      • Ran F.A.
      • Cox D.
      • Lin S.
      • Barretto R.
      • Habib N.
      • Hsu P.D.
      • Wu X.
      • Jiang W.
      • Marraffini L.A.
      • Zhang F.
      Multiplex genome engineering using CRISPR/Cas systems.
      ,
      • Maresca M.
      • Lin V.G.
      • Guo N.
      • Yang Y.
      Obligate ligation-gated recombination (ObLiGaRe): Custom-designed nuclease-mediated targeted integration through nonhomologous end joining.
      ,
      • DeKelver R.C.
      • Choi V.M.
      • Moehle E.A.
      • Paschon D.E.
      • Hockemeyer D.
      • Meijsing S.H.
      • Sancak Y.
      • Cui X.
      • Steine E.J.
      • Miller J.C.
      • Tam P.
      • Bartsevich V.V.
      • Meng X.
      • Rupniewski I.
      • Gopalan S.M.
      • et al.
      Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome.
      ,
      • Ran F.A.
      • Hsu P.D.
      • Lin C.Y.
      • Gootenberg J.S.
      • Konermann S.
      • Trevino A.E.
      • Scott D.A.
      • Inoue A.
      • Matoba S.
      • Zhang Y.
      • Zhang F.
      Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
      ,
      • Maeder M.L.
      • Linder S.J.
      • Cascio V.M.
      • Fu Y.
      • Ho Q.H.
      • Joung J.K.
      CRISPR RNA-guided activation of endogenous human genes.
      ,
      • Chavez A.
      • Scheiman J.
      • Vora S.
      • Pruitt B.W.
      • Tuttle M.
      • Iyer E.P.R.
      • Lin S.
      • Kiani S.
      • Guzman C.D.
      • Wiegand D.J.
      • Ter-Ovanesyan D.
      • Braff J.L.
      • Davidsohn N.
      • Housden B.E.
      • Perrimon N.
      • et al.
      Highly efficient Cas9-mediated transcriptional programming.
      ).

      Targeted KO

      Stable gene KO can be achieved using nuclease-based genome engineering particularly ZFNs, TALENs, and CRISPR/Cas that all can be designed to bind and cleave specific nucleotide sequences and introduce double-strand breaks (DSBs) (
      • Zhang H.X.
      • Zhang Y.
      • Yin H.
      Genome editing with mRNA encoding ZFN, TALEN, and Cas9.
      ,
      • Li H.
      • Yang Y.
      • Hong W.
      • Huang M.
      • Wu M.
      • Zhao X.
      Applications of genome editing technology in the targeted therapy of human diseases: Mechanisms, advances and prospects.
      ). Repair of DSBs through nonhomologous end joining (NHEJ) is error-prone and results in mutations or small insertions/deletions (indels) that disrupt the target gene (
      • Symington L.S.
      • Gautier J.
      Double-strand break end resection and repair pathway choice.
      ) (Fig. 2B). ZFNs and TALENs have very high sequence binding specificity and low off-target effects (
      • Bogdanove A.J.
      • Voytas D.F.
      TAL effectors: Customizable proteins for DNA targeting.
      ); however, design options for target sites are limited and production is laborious, costly and requires expertise although premade targeting constructs are available. CRISPR/Cas gene editing only requires design of guide RNAs (gRNAs), synthetic RNAs with a 16 to 20 nucleotide genomic targeting sequence (crRNA) with a proto-spacer adjacent motif (PAM), and a Cas nuclease-recruiting sequence (tracrRNA) (
      • Jinek M.
      • Chylinski K.
      • Fonfara I.
      • Hauer M.
      • Doudna J.A.
      • Charpentier E.
      A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
      ). Different Cas nucleases exist in different species each recognizing distinct PAMs, and currently this specificity is further engineered to allow broader sequence recognition (
      • Walton R.T.
      • Christie K.A.
      • Whittaker M.N.
      • Kleinstiver B.P.
      Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.
      ). Most CRISPR engineering is based on the class II nucleases Cas9 from S. aureus, to which we refer if not otherwise stated, and more recently Cas12. Established prediction algorithms for optimal candidate gRNA designs are continuously developed; however, candidates still need experimental validation for functional activity (
      • Hsu P.D.
      • Scott D.A.
      • Weinstein J.A.
      • Ran F.A.
      • Konermann S.
      • Agarwala V.
      • Li Y.
      • Fine E.J.
      • Wu X.
      • Shalem O.
      • Cradick T.J.
      • Marraffini L.A.
      • Bao G.
      • Zhang F.
      DNA targeting specificity of RNA-guided Cas9 nucleases.
      ,
      • Doench J.G.
      • Fusi N.
      • Sullender M.
      • Hegde M.
      • Vaimberg E.W.
      • Donovan K.F.
      • Smith I.
      • Tothova Z.
      • Wilen C.
      • Orchard R.
      • Virgin H.W.
      • Listgarten J.
      • Root D.E.
      Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.
      ,
      • Labun K.
      • Montague T.G.
      • Gagnon J.A.
      • Thyme S.B.
      • Valen E.
      CHOPCHOP v2: A web tool for the next generation of CRISPR genome engineering.
      ,
      • Hanna R.E.
      • Doench J.G.
      Design and analysis of CRISPR-Cas experiments.
      ). Libraries of gRNAs for specific targeting of all human glycosyltransferase genes are now available, including a library of validated high efficiency single gRNAs (GlycoCRISPR) (
      • Narimatsu Y.
      • Joshi H.J.
      • Yang Z.
      • Gomes C.
      • Chen Y.H.
      • Lorenzetti F.C.
      • Furukawa S.
      • Schjoldager K.T.
      • Hansen L.
      • Clausen H.
      • Bennett E.P.
      • Wandall H.H.
      A validated gRNA library for CRISPR/Cas9 targeting of the human glycosyltransferase genome.
      ) and a lentiviral viral library containing ten gRNAs (predicted design) per target gene (
      • Zhu Y.
      • Groth T.
      • Kelkar A.
      • Zhou Y.
      • Neelamegham S.
      A GlycoGene CRISPR-Cas9 lentiviral library to study lectin binding and human glycan biosynthesis pathways.
      ). Targeted gene engineering is greatly facilitated by the Indel Detection by Amplicon Analysis assay, which provides single base resolution and informs of the spectrum and frequencies of indels enabling screening for and selecting indels resulting in frameshifts and functional KOs (
      • Bennett E.P.
      • Petersen B.L.
      • Johansen I.E.
      • Niu Y.
      • Yang Z.
      • Chamberlain C.A.
      • Met O.
      • Wandall H.H.
      • Frodin M.
      INDEL detection, the 'achilles heel' of precise genome editing: A survey of methods for accurate profiling of gene editing induced indels.
      ,
      • Lonowski L.A.
      • Narimatsu Y.
      • Riaz A.
      • Delay C.E.
      • Yang Z.
      • Niola F.
      • Duda K.
      • Ober E.A.
      • Clausen H.
      • Wandall H.H.
      • Hansen S.H.
      • Bennett E.P.
      • Frodin M.
      Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis.
      ). Detection of indels may also be based on enzymatic mismatch cleavage assays (
      • Mashal R.D.
      • Koontz J.
      • Sklar J.
      Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases.
      ,
      • Till B.J.
      • Burtner C.
      • Comai L.
      • Henikoff S.
      Mismatch cleavage by single-strand specific nucleases.
      ) and of course different sequencing strategies (
      • Anzalone A.V.
      • Koblan L.W.
      • Liu D.R.
      Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.
      ).

      Targeted KI

      Site-directed and stable introduction of target genes can be achieved by nuclease-based KI. In the presence of a homologous DNA template containing target gene of interest, DSBs can be repaired via homology-directed repair (HDR) resulting in the genomic integration (Fig. 2B). However, HDR occurs at a lower frequency and only in the S/G2 phase compared with the dominant NHEJ repair pathway operative throughout the cell cycle (mainly G1 phase), which makes KI via HDR more challenging (
      • Symington L.S.
      • Gautier J.
      Double-strand break end resection and repair pathway choice.
      ). Therefore, several strategies apply KI independent of the HDR pathway. For example, the obligate ligation-gated recombination (ObLiGaRe) strategy utilizes ZFNs or TALENs to create complementary DNA overhangs (sticky ends) in both the genomic target site and the donor DNA allowing its integration (
      • Maresca M.
      • Lin V.G.
      • Guo N.
      • Yang Y.
      Obligate ligation-gated recombination (ObLiGaRe): Custom-designed nuclease-mediated targeted integration through nonhomologous end joining.
      ,
      • Pinto R.
      • Hansen L.
      • Hintze J.
      • Almeida R.
      • Larsen S.
      • Coskun M.
      • Davidsen J.
      • Mitchelmore C.
      • David L.
      • Troelsen J.T.
      • Bennett E.P.
      Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression.
      ). This KI strategy has already been successfully used for stable KI of GTs (
      • Narimatsu Y.
      • Joshi H.J.
      • Nason R.
      • Van Coillie J.
      • Karlsson R.
      • Sun L.
      • Ye Z.
      • Chen Y.H.
      • Schjoldager K.T.
      • Steentoft C.
      • Furukawa S.
      • Bensing B.A.
      • Sullam P.M.
      • Thompson A.J.
      • Paulson J.C.
      • et al.
      An atlas of human glycosylation pathways enables display of the human glycome by gene engineered cells.
      ,
      • Yang Z.
      • Wang S.
      • Halim A.
      • Schulz M.A.
      • Frodin M.
      • Rahman S.H.
      • Vester-Christensen M.B.
      • Behrens C.
      • Kristensen C.
      • Vakhrushev S.Y.
      • Bennett E.P.
      • Wandall H.H.
      • Clausen H.
      Engineered CHO cells for production of diverse, homogeneous glycoproteins.
      ).
      CRISPR/Cas9-mediated KI utilizes the microhomology-mediated end joining (MMEJ) pathway to repair DSBs (with blunt ends), and foreign DNA flanked with short homologous sequences (5–25 bp) can be used for integration at target sites (
      • Nakade S.
      • Tsubota T.
      • Sakane Y.
      • Kume S.
      • Sakamoto N.
      • Obara M.
      • Daimon T.
      • Sezutsu H.
      • Yamamoto T.
      • Sakuma T.
      • Suzuki K.T.
      Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9.
      ,
      • Suzuki K.
      • Tsunekawa Y.
      • Hernandez-Benitez R.
      • Wu J.
      • Zhu J.
      • Kim E.J.
      • Hatanaka F.
      • Yamamoto M.
      • Araoka T.
      • Li Z.
      • Kurita M.
      • Hishida T.
      • Li M.
      • Aizawa E.
      • Guo S.
      • et al.
      In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.
      ,
      • Sakuma T.
      • Nakade S.
      • Sakane Y.
      • Suzuki K.T.
      • Yamamoto T.
      MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems.
      ). CRISPR strategies have improved KI efficiency via HDR by virtue of Cas9 nickases (Cas9n), Cas9 nucleases with an inactivating mutation in either the HNH (Cas9H840A) or RuvC (Cas9D10A) domain (
      • Cong L.
      • Ran F.A.
      • Cox D.
      • Lin S.
      • Barretto R.
      • Habib N.
      • Hsu P.D.
      • Wu X.
      • Jiang W.
      • Marraffini L.A.
      • Zhang F.
      Multiplex genome engineering using CRISPR/Cas systems.
      ,
      • Ran F.A.
      • Hsu P.D.
      • Lin C.Y.
      • Gootenberg J.S.
      • Konermann S.
      • Trevino A.E.
      • Scott D.A.
      • Inoue A.
      • Matoba S.
      • Zhang Y.
      • Zhang F.
      Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
      ,
      • Nishimasu H.
      • Ran F.A.
      • Hsu P.D.
      • Konermann S.
      • Shehata S.I.
      • Dohmae N.
      • Ishitani R.
      • Zhang F.
      • Nureki O.
      Crystal structure of Cas9 in complex with guide RNA and target DNA.
      ). Cas9n creates single-strand breaks (nicks), and by pairing two nickases and two gRNAs for both DNA strands, DSBs with long overhangs can be created. Donor DNA flanked with complementary arms or simultaneously nicked donor DNA can then be inserted at the DSB site via HDR or non-HDR mechanisms with high efficiency and low off-target effects (
      • Ran F.A.
      • Hsu P.D.
      • Lin C.Y.
      • Gootenberg J.S.
      • Konermann S.
      • Trevino A.E.
      • Scott D.A.
      • Inoue A.
      • Matoba S.
      • Zhang Y.
      • Zhang F.
      Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
      ,
      • Chen X.
      • Janssen J.M.
      • Liu J.
      • Maggio I.
      • t Jong A.E.J.
      • Mikkers H.M.M.
      • Goncalves M.
      In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting.
      ,
      • Koch B.
      • Nijmeijer B.
      • Kueblbeck M.
      • Cai Y.
      • Walther N.
      • Ellenberg J.
      Generation and validation of homozygous fluorescent knock-in cells using CRISPR-Cas9 genome editing.
      ,
      • Goncalves M.A.
      • van Nierop G.P.
      • Holkers M.
      • de Vries A.A.
      Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells.
      ). To overcome random integration of artificial DNA sequences into the genome and off-target effects, the AAVS1 locus is often used as safe-harbor site for KI (
      • DeKelver R.C.
      • Choi V.M.
      • Moehle E.A.
      • Paschon D.E.
      • Hockemeyer D.
      • Meijsing S.H.
      • Sancak Y.
      • Cui X.
      • Steine E.J.
      • Miller J.C.
      • Tam P.
      • Bartsevich V.V.
      • Meng X.
      • Rupniewski I.
      • Gopalan S.M.
      • et al.
      Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome.
      ,
      • Papapetrou E.P.
      • Schambach A.
      Gene insertion into genomic safe harbors for human gene therapy.
      ). Gene KI can make use of customizable constructs for constitutively active expression of target genes, for example, with or without tags and also holds the opportunity to insert tunable expression constructs, for example, under control of the Tet-On/Off system, and this has already been applied for glycoengineering (
      • Hintze J.
      • Ye Z.
      • Narimatsu Y.
      • Madsen T.D.
      • Joshi H.J.
      • Goth C.K.
      • Linstedt A.
      • Bachert C.
      • Mandel U.
      • Bennett E.P.
      • Vakhrushev S.Y.
      • Schjoldager K.T.
      Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines.
      ).

      Regulating endogenous gene expression

      The CRISPR technology is rapidly evolving by adding functionalities to Cas nucleases. Utilization of catalytically inactive (dead) dCas9 allows to fully repress target gene expression through fusion with transcription repressors (CRISPR interference, CRISPRi) and recruitment to the promoter region of a target gene (
      • Gilbert L.A.
      • Larson M.H.
      • Morsut L.
      • Liu Z.
      • Brar G.A.
      • Torres S.E.
      • Stern-Ginossar N.
      • Brandman O.
      • Whitehead E.H.
      • Doudna J.A.
      • Lim W.A.
      • Weissman J.S.
      • Qi L.S.
      CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.
      ). Likewise, fusion with transcriptional activators such as VP64, p65, and Rta enables activation of gene expression (CRISPR activation, CRISPRa) (
      • Maeder M.L.
      • Linder S.J.
      • Cascio V.M.
      • Fu Y.
      • Ho Q.H.
      • Joung J.K.
      CRISPR RNA-guided activation of endogenous human genes.
      ,
      • Chavez A.
      • Scheiman J.
      • Vora S.
      • Pruitt B.W.
      • Tuttle M.
      • Iyer E.P.R.
      • Lin S.
      • Kiani S.
      • Guzman C.D.
      • Wiegand D.J.
      • Ter-Ovanesyan D.
      • Braff J.L.
      • Davidsohn N.
      • Housden B.E.
      • Perrimon N.
      • et al.
      Highly efficient Cas9-mediated transcriptional programming.
      ,
      • Matharu N.
      • Rattanasopha S.
      • Tamura S.
      • Maliskova L.
      • Wang Y.
      • Bernard A.
      • Hardin A.
      • Eckalbar W.L.
      • Vaisse C.
      • Ahituv N.
      CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency.
      ) (Fig. 2B). CRISPRa was applied to activate the fucosyltransferase 4 and 9 genes (fut4 and fut9) in a murine cell line resulting in Lewisx expression (
      • Blanas A.
      • Cornelissen L.A.M.
      • Kotsias M.
      • van der Horst J.C.
      • van de Vrugt H.J.
      • Kalay H.
      • Spencer D.I.R.
      • Kozak R.P.
      • van Vliet S.J.
      Transcriptional activation of fucosyltransferase (FUT) genes using the CRISPR-dCas9-VPR technology reveals potent N-glycome alterations in colorectal cancer cells.
      ). Other CRISPR-based approaches for gene regulation have emerged that are potentially useful for glycoengineering. For example, dCas9 fused to base editors such as cytidine deaminase that coverts cytidine to uridine allows introduction of targeted point mutations (base editing), fusion to a reverse transcriptase domain for prime editing, fusion to epigenetic modifiers (e.g., P300, TET1, LSD1, DNMT3A) enables epigenome editing, RNA-targeting Cas13a nuclease enables posttranscriptional engineering of mRNA, and CRISPR tools are emerging for mutagenesis and directed evolution (
      • Doudna J.A.
      The promise and challenge of therapeutic genome editing.
      ,
      • Anzalone A.V.
      • Koblan L.W.
      • Liu D.R.
      Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.
      ,
      • Molla K.A.
      • Yang Y.
      CRISPR/Cas-mediated base editing: Technical considerations and practical applications.
      ,
      • Knott G.J.
      • Doudna J.A.
      CRISPR-Cas guides the future of genetic engineering.
      ,
      • Katayama K.
      • Mitsunobu H.
      • Nishida K.
      Mammalian synthetic biology by CRISPRs engineering and applications.
      ). Currently, the main obstacle for extensive glycoengineering is limitations in introducing multiple genes by targeted KI, and this obstacle may be alleviated by activation of endogenous nonexpressed genes. Another interesting opportunity for engineering is to tag endogenous GTs or their substrate proteins to enable selective monitoring of expression of enzymes or their effects on specific proteins in live cells (
      • Schmid-Burgk J.L.
      • Honing K.
      • Ebert T.S.
      • Hornung V.
      CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism.
      ).

      Delivery gene editing

      A key step in genetic engineering is the delivery of editing components such as DNA, RNA, and/or protein (
      • van Haasteren J.
      • Li J.
      • Scheideler O.J.
      • Murthy N.
      • Schaffer D.V.
      The delivery challenge: Fulfilling the promise of therapeutic genome editing.
      ,
      • Lino C.A.
      • Harper J.C.
      • Carney J.P.
      • Timlin J.A.
      Delivering CRISPR: A review of the challenges and approaches.
      ) (Fig. 2B). DNA plasmids are most commonly used, because of ease of availability and use, stability, and flexibility in design, but genomic insertions of plasmids may lead to off-target effects. Use of RNA circumvents this as RNA is readily translated in the cytoplasm resulting in fast protein production, and transfecting with RNA has advantages especially with primary and difficult-to-transfect cells (
      • Oh S.
      • Kessler J.A.
      Design, assembly, production, and transfection of synthetic modified mRNA.
      ). However, RNA is more costly and difficult to handle, unstable, less customizable, and expression of the encoded protein is lower and relatively short-lived. The Cas9 protein may be delivered precomplexed with gRNAs and used directly in transfection allowing for control over dosage, and this may be effective with cells difficult to transfect with DNA or RNA, but requires highly pure protein and protocol optimization for different proteins. The methods of delivery include chemical, biological, or physical means, and the efficacy of these varies greatly (
      • Doudna J.A.
      The promise and challenge of therapeutic genome editing.
      ,
      • van Haasteren J.
      • Li J.
      • Scheideler O.J.
      • Murthy N.
      • Schaffer D.V.
      The delivery challenge: Fulfilling the promise of therapeutic genome editing.
      ,
      • Lino C.A.
      • Harper J.C.
      • Carney J.P.
      • Timlin J.A.
      Delivering CRISPR: A review of the challenges and approaches.
      ). For example, the human embryonic kidney (HEK293) cell is readily transfectable with cost-effective chemical reagents such as calcium phosphate, polyethylenimine (PEI) polymers, and liposomes. More difficult-to-transfect cells such as primary cells and nondividing cells may be efficiently transfected by electroporation, nucleofection, or other methods that temporarily induce pores in the plasma membrane; however, these often result in varying levels of cytotoxicity (
      • Doudna J.A.
      The promise and challenge of therapeutic genome editing.
      ,
      • Dever D.P.
      • Bak R.O.
      • Reinisch A.
      • Camarena J.
      • Washington G.
      • Nicolas C.E.
      • Pavel-Dinu M.
      • Saxena N.
      • Wilkens A.B.
      • Mantri S.
      • Uchida N.
      • Hendel A.
      • Narla A.
      • Majeti R.
      • Weinberg K.I.
      • et al.
      CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells.
      ). Lentiviral transduction may be the option of choice for difficult-to-transfect cells and enables stable integration of highly customizable vectors. This was applied, e.g., for glycoengineering of human keratinocytes to develop organotypic skin models (
      • Dabelsteen S.
      • Pallesen E.M.H.
      • Marinova I.N.
      • Nielsen M.I.
      • Adamopoulou M.
      • Romer T.B.
      • Levann A.
      • Andersen M.M.
      • Ye Z.
      • Thein D.
      • Bennett E.P.
      • Bull C.
      • Moons S.J.
      • Boltje T.
      • Clausen H.
      • et al.
      Essential functions of glycans in human epithelia dissected by a CRISPR-cas9-engineered human organotypic skin model.
      ), and a comprehensive lentiviral glycogene CRISPR library has been designed (
      • Zhu Y.
      • Groth T.
      • Kelkar A.
      • Zhou Y.
      • Neelamegham S.
      A GlycoGene CRISPR-Cas9 lentiviral library to study lectin binding and human glycan biosynthesis pathways.
      ). Notably, level and timing of Cas9 expression are difficult to control with these methods and high prolonged expression enhances off-target frequencies (
      • Lonowski L.A.
      • Narimatsu Y.
      • Riaz A.
      • Delay C.E.
      • Yang Z.
      • Niola F.
      • Duda K.
      • Ober E.A.
      • Clausen H.
      • Wandall H.H.
      • Hansen S.H.
      • Bennett E.P.
      • Frodin M.
      Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis.
      ). This hurdle may be overcome by inactivating CRISPR systems that allow controlled and temporal expression levels of Cas9, and these have already been applied to reduce off-target effects of editing the human α3-sialyltransferase 4 gene (ST3GAL4) (
      • Kelkar A.
      • Zhu Y.
      • Groth T.
      • Stolfa G.
      • Stablewski A.B.
      • Singhi N.
      • Nemeth M.
      • Neelamegham S.
      Doxycycline-dependent self-inactivation of CRISPR-cas9 to temporally regulate on- and off-target editing.
      ). Selection markers such as antibiotics or fluorescent reporters, e.g., GFP-tagged Cas9, facilitate enrichment of transfected cells with desired nuclease expression levels. Fluorescence activated cell sorting (FACS) is useful to select and enrich for cells with desirable expression of GFP-tagged Cas9 (
      • Lonowski L.A.
      • Narimatsu Y.
      • Riaz A.
      • Delay C.E.
      • Yang Z.
      • Niola F.
      • Duda K.
      • Ober E.A.
      • Clausen H.
      • Wandall H.H.
      • Hansen S.H.
      • Bennett E.P.
      • Frodin M.
      Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis.
      ,
      • Ren C.
      • Xu K.
      • Segal D.J.
      • Zhang Z.
      Strategies for the enrichment and selection of genetically modified cells.
      ), as is further selection of clones with desirable changes in glycosylation and/or expression of targeted proteins by lectins and antibodies.

      Engineering of glycosylation capacities in cells

      Two conceptionally opposing strategies for glycoengineering have been applied. One approach takes origin in glycan structures and seeks to identify genes required for biosynthesis and expression of specific glycans—here designated as random glycoengineering. The other approach takes origin in the glycosylation capacities of a cell and seeks to engineer the repertoire of expressed GTs to accommodate biosynthesis of glycans of interest—here designated as rational glycoengineering. These strategies are not mutually exclusive.

      Random glycoengineering—discovery potential

      Random glycoengineering was pioneered by Stanley et al. who generated glycosylation mutants in Chinese hamster ovary (CHO) cells, designated Lec or LEC (for loss-of-function and gain-of-function mutants, respectively). CHO cells were subjected to random mutagenesis followed by positive or negative selection with cytotoxic plant or bacterial lectins to obtain lectin-resistant mutants with loss/gain of a glycan feature. With this approach a large number of valuable CHO glycosylation mutants were isolated leading to the discovery and characterization of GTs, sugar nucleotide transporters, and other genes (
      • Patnaik S.K.
      • Stanley P.
      Lectin-resistant CHO glycosylation mutants.
      ,
      • Esko J.D.
      • Stanley P.
      Glycosylation mutants of cultured mammalian cells.
      ). Later, more systematic studies with random insertional mutagenesis using gene trapping in haploid HAP1 cells and selection with Lassa virus binding to the matriglycan on α-dystroglycan revealed several known (e.g., POMT1/2, B3GNT1) and unknown (e.g., SLC35A1, ST3GAL4, B3GALNT2) glycogenes involved in glycosylation of α-dystroglycan and underlying dystroglycanopathies (
      • Jae L.T.
      • Raaben M.
      • Riemersma M.
      • van Beusekom E.
      • Blomen V.A.
      • Velds A.
      • Kerkhoven R.M.
      • Carette J.E.
      • Topaloglu H.
      • Meinecke P.
      • Wessels M.W.
      • Lefeber D.J.
      • Whelan S.P.
      • van Bokhoven H.
      • Brummelkamp T.R.
      Deciphering the glycosylome of dystroglycanopathies using haploid screens for lassa virus entry.
      ). Random mutagenesis studies are now being substituted by whole genome screens (or select gene subsets) using CRISPR libraries (
      • Kampmann M.
      CRISPR-based functional genomics for neurological disease.
      ,
      • Weber J.
      • Braun C.J.
      • Saur D.
      • Rad R.
      In vivo functional screening for systems-level integrative cancer genomics.
      ). These and other studies have uncovered unexpected genes that direct or regulate cellular glycosylation processes in interactions with bacterial toxins, viral infections, and other molecular events as summarized in Table 1. This strategy relies on the efficiency of CRISPR/Cas targeting in introducing biallelic indels (and multiallelic) in target genes necessary to induce complete loss of function of GT enzyme activities and thus partly omitting the benefits of using haploid cell lines where mainly a single allele of genes requires targeting. Technical limitations of genome-wide CRISPR/Cas KO screens are off-target effects and false-negative hits caused by nondeleterious indels (
      • Kampmann M.
      CRISPR-based functional genomics for neurological disease.
      ,
      • Weber J.
      • Braun C.J.
      • Saur D.
      • Rad R.
      In vivo functional screening for systems-level integrative cancer genomics.
      ), but also a low likelihood of identifying GTs that are members of partially redundant isoenzyme families (
      • Schjoldager K.T.
      • Narimatsu Y.
      • Joshi H.J.
      • Clausen H.
      Global view of human protein glycosylation pathways and functions.
      ). Thus, the CRISPR/Cas screening strategy has mainly identified nonredundant genes with global effects on glycosylation (Table 1). For example, genome-wide screening for genes required for avian influenza virus replication identified the Golgi CMP–sialic acid transporter (SLC35A1) and confirming requirement for sialic acid receptors; however, none of the many sialyltransferase genes necessary for sialylation were identified (
      • Han J.
      • Perez J.T.
      • Chen C.
      • Li Y.
      • Benitez A.
      • Kandasamy M.
      • Lee Y.
      • Andrade J.
      • ten Oever B.
      • Manicassamy B.
      Genome-wide CRISPR/Cas9 screen identifies host factors essential for influenza virus replication.
      ). A genome-wide CRISPR screen for hepatitis A virus entry identified multiple genes in the ganglioside biosynthetic pathway, including UDP-glucose ceramide glucosyltransferase (UGCG) and β4-galactosyltransferase 5 (B4GALT5) directing synthesis of Galβ1-4Glcβ1-Cer, and the lactosylceramide α3-sialyltransferase (ST3GAL5), which may only serve as the GM3 synthase (NeuAcα2-3Galβ1-4Glcβ1-Cer) (
      • Das A.
      • Barrientos R.
      • Shiota T.
      • Madigan V.
      • Misumi I.
      • McKnight K.L.
      • Sun L.
      • Li Z.
      • Meganck R.M.
      • Li Y.
      • Kaluzna E.
      • Asokan A.
      • Whitmire J.K.
      • Kapustina M.
      • Zhang Q.
      • et al.
      Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus.
      ). Multiple genome-wide screens have shown the importance of N-glycosylation and the OST complex for Dengue virus infection (
      • Lin D.L.
      • Cherepanova N.A.
      • Bozzacco L.
      • MacDonald M.R.
      • Gilmore R.
      • Tai A.W.
      Dengue virus hijacks a noncanonical oxidoreductase function of a cellular oligosaccharyltransferase complex.
      ,
      • Marceau C.D.
      • Puschnik A.S.
      • Majzoub K.
      • Ooi Y.S.
      • Brewer S.M.
      • Fuchs G.
      • Swaminathan K.
      • Mata M.A.
      • Elias J.E.
      • Sarnow P.
      • Carette J.E.
      Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens.
      ,
      • Ooi Y.S.
      • Majzoub K.
      • Flynn R.A.
      • Mata M.A.
      • Diep J.
      • Li J.K.
      • van Buuren N.
      • Rumachik N.
      • Johnson A.G.
      • Puschnik A.S.
      • Marceau C.D.
      • Mlera L.
      • Grabowski J.M.
      • Kirkegaard K.
      • Bloom M.E.
      • et al.
      An RNA-centric dissection of host complexes controlling flavivirus infection.
      ), and one screen in the HAP1 haploid cell line also identified multiple genes in the heparan sulfate pathway indicating a role of GAGs as well (
      • Labeau A.
      • Simon-Loriere E.
      • Hafirassou M.L.
      • Bonnet-Madin L.
      • Tessier S.
      • Zamborlini A.
      • Dupre T.
      • Seta N.
      • Schwartz O.
      • Chaix M.L.
      • Delaugerre C.
      • Amara A.
      • Meertens L.
      A genome-wide CRISPR-cas9 screen identifies the dolichol-phosphate mannose synthase complex as a host dependency factor for dengue virus infection.
      ).
      Table 1Genome-wide screens in mammalian cells identifying genes involved in glycosylation
      Cell typeGenome-wide screenPhenotypic selectionIdentified glycogenesReference
      Chronic myeloid leukemia cells HAP1 (haploid)Gene trap mutagenesisResistance to Lassa virus entry via α-dystroglycanPOMT1/2, B3GNT1, SLC35A1, ST3GAL4, B3GALNT2(
      • Jae L.T.
      • Raaben M.
      • Riemersma M.
      • van Beusekom E.
      • Blomen V.A.
      • Velds A.
      • Kerkhoven R.M.
      • Carette J.E.
      • Topaloglu H.
      • Meinecke P.
      • Wessels M.W.
      • Lefeber D.J.
      • Whelan S.P.
      • van Bokhoven H.
      • Brummelkamp T.R.
      Deciphering the glycosylome of dystroglycanopathies using haploid screens for lassa virus entry.
      )
      Human cervical cancer cells HeLaCRISPR/Cas9 KO screen
      Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      Resistance to Shiga toxin (Stx)UGCG, B4GALT5, SPTLC2, A4GALT, SPTLC1(
      • Majumder S.
      • Kono M.
      • Lee Y.T.
      • Byrnes C.
      • Li C.
      • Tuymetova G.
      • Proia R.L.
      A genome-wide CRISPR/Cas9 screen reveals that the aryl hydrocarbon receptor stimulates sphingolipid levels.
      )
      Human cervical cancer cells HeLaCRISPR/Cas9 KO screen
      Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      Resistance to Shiga toxin (Stx)A4GALT, B4GALT5, UGCG, GALE, SLC35A2(
      • Yamaji T.
      • Sekizuka T.
      • Tachida Y.
      • Sakuma C.
      • Morimoto K.
      • Kuroda M.
      • Hanada K.
      A CRISPR screen identifies LAPTM4A and TM9SF proteins as glycolipid-regulating factors.
      )
      Human bladder cancer cells 5637CRISPR/Cas9 KO screen
      Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      Resistance to Shiga-like toxins (Stxs) 1 and 2A4GALT, B4GALT5, SLC35A2, UGCG, SLC35A2(
      • Tian S.
      • Muneeruddin K.
      • Choi M.Y.
      • Tao L.
      • Bhuiyan R.H.
      • Ohmi Y.
      • Furukawa K.
      • Furukawa K.
      • Boland S.
      • Shaffer S.A.
      • Adam R.M.
      • Dong M.
      Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation.
      )
      Human colorectal adenocarcinoma cells HT-29CRISPR/Cas9 KO screen
      Lentiviral AVANA sgRNA library targeting 18,675 genes.
      Resistance to EHEC cytotoxicity (T3SS, Stx1 and Stx2)A4GALT, B4GALT5, UGCG(
      • Pacheco A.R.
      • Lazarus J.E.
      • Sit B.
      • Schmieder S.
      • Lencer W.I.
      • Blondel C.J.
      • Doench J.G.
      • Davis B.M.
      • Waldor M.K.
      CRISPR screen reveals that EHEC's T3SS and Shiga toxin rely on shared host factors for infection.
      )
      Human cervical cancer cells HeLaCRISPR/Cas9 KO screen
      Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      Resistance to ricin toxinALG5, ALG6, ALG8, MOGS, OST4, MAN1A2, MAN2A1, MGAT1, MGAT2, SLC35C1, FUT4(
      • Tian S.
      • Muneeruddin K.
      • Choi M.Y.
      • Tao L.
      • Bhuiyan R.H.
      • Ohmi Y.
      • Furukawa K.
      • Furukawa K.
      • Boland S.
      • Shaffer S.A.
      • Adam R.M.
      • Dong M.
      Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation.
      )
      Human cervical cancer cells HeLaCRISPR/Cas9 KO screen
      Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      Resistance to E. coli subtilase cytotoxin (SubAB)-induced cell deathSLC39A9, CMAS, SLC35A1, MGAT1, C1GALT1, C1GALT1C1(
      • Yamaji T.
      • Hanamatsu H.
      • Sekizuka T.
      • Kuroda M.
      • Iwasaki N.
      • Ohnishi M.
      • Furukawa J.I.
      • Yahiro K.
      • Hanada K.
      A CRISPR screen using subtilase cytotoxin identifies SLC39A9 as a glycan-regulating factor.
      )
      Human cervical cancer cells HeLaCRISPR/Cas9 KO screen
      Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      Gal-3 cell surface localizationSLC35A2, MGAT1, MAN1A2, SLC39A9(
      • Stewart S.E.
      • Menzies S.A.
      • Popa S.J.
      • Savinykh N.
      • Petrunkina Harrison A.
      • Lehner P.J.
      • Moreau K.
      A genome-wide CRISPR screen reconciles the role of N-linked glycosylation in galectin-3 transport to the cell surface.
      )
      Human hepatocellular carcinoma cells Huh7.5.1CRISPR/Cas9 KO screen
      Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      Resistance to Ebola virus infectionSLC30A1, GNPTAB(
      • Flint M.
      • Chatterjee P.
      • Lin D.L.
      • McMullan L.K.
      • Shrivastava-Ranjan P.
      • Bergeron E.
      • Lo M.K.
      • Welch S.R.
      • Nichol S.T.
      • Tai A.W.
      • Spiropoulou C.F.
      A genome-wide CRISPR screen identifies N-acetylglucosamine-1-phosphate transferase as a potential antiviral target for Ebola virus.
      )
      Human cervical cancer cells HeLa and human embryonic kidney cells HEK293CRISPR/Cas9 KO screen
      Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      Resistance to West Nile virus infectionSTT3A, OST4, OSTC(
      • Zhang R.
      • Miner J.J.
      • Gorman M.J.
      • Rausch K.
      • Ramage H.
      • White J.P.
      • Zuiani A.
      • Zhang P.
      • Fernandez E.
      • Zhang Q.
      • Dowd K.A.
      • Pierson T.C.
      • Cherry S.
      • Diamond M.S.
      A CRISPR screen defines a signal peptide processing pathway required by flaviviruses.
      )
      Chronic myeloid leukemia cells HAP1 (haploid)CRISPR/Cas9 KO screen
      Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      Resistance to Dengue virus infectionSTT3A, STT3B, RPN2, B4GALT7, B3GALT6, B3GAT3, PAPSS1, SLC35B2

      DPM1, DPM3
      (
      • Labeau A.
      • Simon-Loriere E.
      • Hafirassou M.L.
      • Bonnet-Madin L.
      • Tessier S.
      • Zamborlini A.
      • Dupre T.
      • Seta N.
      • Schwartz O.
      • Chaix M.L.
      • Delaugerre C.
      • Amara A.
      • Meertens L.
      A genome-wide CRISPR-cas9 screen identifies the dolichol-phosphate mannose synthase complex as a host dependency factor for dengue virus infection.
      )
      Chronic myeloid leukemia cells HAP1 (haploid) and Human hepatocellular carcinoma cells Huh7.5.1CRISPR/Cas9 KO screen
      Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      Resistance to Dengue virus infectionSTT3A, STT3B, MGAT1, OSTC, RPN1,RPN2, OST4, B3GALT6, EXT1(
      • Marceau C.D.
      • Puschnik A.S.
      • Majzoub K.
      • Ooi Y.S.
      • Brewer S.M.
      • Fuchs G.
      • Swaminathan K.
      • Mata M.A.
      • Elias J.E.
      • Sarnow P.
      • Carette J.E.
      Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens.
      )
      Human lung epithelial cells A549CRISPR/Cas9 KO screen
      Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      Resistance to IAV (H5N1) virus infectionSLC35A1, DPM2, ALG3, ALG4, ALG12, GANAB, A4GALT, B3GAT1, B4GALNT4, CHSY1, CSGALNACT2, HS3ST6, PIGN, DPM2(
      • Han J.
      • Perez J.T.
      • Chen C.
      • Li Y.
      • Benitez A.
      • Kandasamy M.
      • Lee Y.
      • Andrade J.
      • ten Oever B.
      • Manicassamy B.
      Genome-wide CRISPR/Cas9 screen identifies host factors essential for influenza virus replication.
      )
      Human lung epithelial cells A549CRISPR/dCas9 activation screen
      Lentiviral sgRNA library targeting upstream TSS of 23,430 coding isoforms.
      Blocking IAV (H1N1/PR8/1934) infectionB4GALNT2(
      • Heaton B.E.
      • Kennedy E.M.
      • Dumm R.E.
      • Harding A.T.
      • Sacco M.T.
      • Sachs D.
      • Heaton N.S.
      A CRISPR activation screen identifies a pan-avian influenza virus inhibitory host factor.
      )
      Human colorectal adenocarcinoma cells HT-29CRISPR/Cas9 KO screen
      Lentiviral AVANA sgRNA library targeting 18,675 genes.
      Resistance to V. parahaemolyticus cytotoxicity (T3SS1 and T3SS2)SLC35B2, SLC35B3, HS6ST1, SLC35C1, GMD, FUT4, SLC35A2(
      • Blondel C.J.
      • Park J.S.
      • Hubbard T.P.
      • Pacheco A.R.
      • Kuehl C.J.
      • Walsh M.J.
      • Davis B.M.
      • Gewurz B.E.
      • Doench J.G.
      • Waldor M.K.
      CRISPR/Cas9 screens reveal requirements for host cell sulfation and fucosylation in bacterial type III secretion system-mediated cytotoxicity.
      )
      Human lymphoma cells JeKo-1CRISPR/dCas9 activation screen
      Lentiviral sgRNA library targeting upstream TSS of 23,430 coding isoforms.
      Resistance to anti/CD3xCD20 bispecific antibody-mediated killingB4GALNT1, B3GNT4(
      • Decker C.E.
      • Young T.
      • Pasnikowski E.
      • Chiu J.
      • Song H.
      • Wei Y.
      • Thurston G.
      • Daly C.
      Genome-scale CRISPR activation screen uncovers tumor-intrinsic modulators of CD3 bispecific antibody efficacy.
      )
      a Lentiviral GeCKO sgRNA library targeting 19,050 genes and 1864 miRNAs.
      b Lentiviral AVANA sgRNA library targeting 18,675 genes.
      c Lentiviral sgRNA library targeting upstream TSS of 23,430 coding isoforms.
      Genes that are not endogenously expressed in the cell line used for screening are of course disregarded, but this limitation may partly be overcome with the emerging genome-wide CRISPR activation libraries that enable gain-of-function screening. For example, a genome-wide CRISPR/dCas9 screen for inhibitory factors of α2-3Sia binding influenza A virus uncovered β4-GalNAc-transferase 2 (B4GALNT2) and revealed that addition of a β4GalNAc residue to α2-3Sia capped glycans to form the SDa epitope (Neu5Acα2-3[GalNAcβ1-4]Galβ1-R) blocks binding (
      • Heaton B.E.
      • Kennedy E.M.
      • Dumm R.E.
      • Harding A.T.
      • Sacco M.T.
      • Sachs D.
      • Heaton N.S.
      A CRISPR activation screen identifies a pan-avian influenza virus inhibitory host factor.
      ). The unbiased nature of random engineering studies has demonstrated great potential for discoveries of GT genes and their functions in cellular glycosylation processes.

      Rational genetic glycoengineering—custom design and dissection

      Rational glycoengineering represents a systematic and versatile strategy for studying and dissecting biological roles of glycosylation. Advanced understanding of the genetic and biosynthetic circuits of the cellular glycosylation machinery and regulation of the glycome (Fig. 2A) (
      • Lowe J.B.
      • Marth J.D.
      A genetic approach to mammalian glycan function.
      ,
      • Schjoldager K.T.
      • Narimatsu Y.
      • Joshi H.J.
      • Clausen H.
      Global view of human protein glycosylation pathways and functions.
      ,
      • Henrissat B.
      • Surolia A.
      • Stanley P.
      A genomic view of glycobiology.
      ,
      • Joshi H.J.
      • Hansen L.
      • Narimatsu Y.
      • Freeze H.H.
      • Henrissat B.
      • Bennett E.
      • Wandall H.H.
      • Clausen H.
      • Schjoldager K.T.
      Glycosyltransferase genes that cause monogenic congenital disorders of glycosylation are distinct from glycosyltransferase genes associated with complex diseases.
      ), combined with the facile gene-editing technologies and libraries of validated gRNAs such as the GlycoCRISPR resource (
      • Narimatsu Y.
      • Joshi H.J.
      • Yang Z.
      • Gomes C.
      • Chen Y.H.
      • Lorenzetti F.C.
      • Furukawa S.
      • Schjoldager K.T.
      • Hansen L.
      • Clausen H.
      • Bennett E.P.
      • Wandall H.H.
      A validated gRNA library for CRISPR/Cas9 targeting of the human glycosyltransferase genome.
      ), is enabling wide use of rational glycoengineering by KO/KI of genes. There appears to be few restrictions to the extent of reprogramming of glycosylation possible in cell lines, and extensive engineering of a majority of the known GT genes has been performed in mammalian cells (predominantly CHO and HEK293) (
      • Narimatsu Y.
      • Joshi H.J.
      • Nason R.
      • Van Coillie J.
      • Karlsson R.
      • Sun L.
      • Ye Z.
      • Chen Y.H.
      • Schjoldager K.T.
      • Steentoft C.
      • Furukawa S.
      • Bensing B.A.
      • Sullam P.M.
      • Thompson A.J.
      • Paulson J.C.
      • et al.
      An atlas of human glycosylation pathways enables display of the human glycome by gene engineered cells.
      ,
      • Yang Z.
      • Wang S.
      • Halim A.
      • Schulz M.A.
      • Frodin M.
      • Rahman S.H.
      • Vester-Christensen M.B.
      • Behrens C.
      • Kristensen C.
      • Vakhrushev S.Y.
      • Bennett E.P.
      • Wandall H.H.
      • Clausen H.
      Engineered CHO cells for production of diverse, homogeneous glycoproteins.
      ,
      • Dabelsteen S.
      • Pallesen E.M.H.
      • Marinova I.N.
      • Nielsen M.I.
      • Adamopoulou M.
      • Romer T.B.
      • Levann A.
      • Andersen M.M.
      • Ye Z.
      • Thein D.
      • Bennett E.P.
      • Bull C.
      • Moons S.J.
      • Boltje T.
      • Clausen H.
      • et al.
      Essential functions of glycans in human epithelia dissected by a CRISPR-cas9-engineered human organotypic skin model.
      ,
      • Narimatsu Y.
      • Joshi H.J.
      • Schjoldager K.T.
      • Hintze J.
      • Halim A.
      • Steentoft C.
      • Nason R.
      • Mandel U.
      • Bennett E.P.
      • Clausen H.
      • Vakhrushev S.Y.
      Exploring regulation of protein O-glycosylation in isogenic human HEK293 cells by differential O-glycoproteomics.
      ,
      • Chen Y.H.
      • Narimatsu Y.
      • Clausen T.M.
      • Gomes C.
      • Karlsson R.
      • Steentoft C.
      • Spliid C.B.
      • Gustavsson T.
      • Salanti A.
      • Persson A.
      • Malmstrom A.
      • Willen D.
      • Ellervik U.
      • Bennett E.P.
      • Mao Y.
      • et al.
      The GAGOme: A cell-based library of displayed glycosaminoglycans.
      ,
      • Tian W.
      • Ye Z.
      • Wang S.
      • Schulz M.A.
      • Van Coillie J.
      • Sun L.
      • Chen Y.H.
      • Narimatsu Y.
      • Hansen L.
      • Kristensen C.
      • Mandel U.
      • Bennett E.P.
      • Jabbarzadeh-Tabrizi S.
      • Schiffmann R.
      • Shen J.S.
      • et al.
      The glycosylation design space for recombinant lysosomal replacement enzymes produced in CHO cells.
      ,
      • Stopschinski B.E.
      • Holmes B.B.
      • Miller G.M.
      • Manon V.A.
      • Vaquer-Alicea J.
      • Prueitt W.L.
      • Hsieh-Wilson L.C.
      • Diamond M.I.
      Specific glycosaminoglycan chain length and sulfation patterns are required for cell uptake of tau versus alpha-synuclein and beta-amyloid aggregates.
      ,
      • Qiu H.
      • Shi S.
      • Yue J.
      • Xin M.
      • Nairn A.V.
      • Lin L.
      • Liu X.
      • Li G.
      • Archer-Hartmann S.A.
      • Dela Rosa M.
      • Galizzi M.
      • Wang S.
      • Zhang F.
      • Azadi P.
      • van Kuppevelt T.H.
      • et al.
      A mutant-cell library for systematic analysis of heparan sulfate structure-function relationships.
      ). Thus, it is now essentially possible to consider the blueprint of glycosylation pathways (Fig. 2A) and deconstruct and/or reconstruct any of these without substantially affecting cell viability and performance, or, for example, expression and secretion of recombinant glycoproteins (
      • Tian W.
      • Ye Z.
      • Wang S.
      • Schulz M.A.
      • Van Coillie J.
      • Sun L.
      • Chen Y.H.
      • Narimatsu Y.
      • Hansen L.
      • Kristensen C.
      • Mandel U.
      • Bennett E.P.
      • Jabbarzadeh-Tabrizi S.
      • Schiffmann R.
      • Shen J.S.
      • et al.
      The glycosylation design space for recombinant lysosomal replacement enzymes produced in CHO cells.
      ). Rational genetic engineering may also apply to the many enzymatic modifications of glycans including epimerization and attachment of sulfate, phosphate, acetyl, methyl, and other groups (
      • Cummings R.D.
      The repertoire of glycan determinants in the human glycome.
      ). The glycome is further shaped by endogenous glycoside hydrolases and in particularly the four mammalian neuraminidases (NEU1–4) may affect the degree of sialic acid capping (
      • Monti E.
      • Miyagi T.
      Structure and function of mammalian sialidases.
      ). Design of rational glycoengineering experiments needs to consider the enzymes expressed in the cell of choice and potential genetic redundancy for biosynthetic steps provided by isoenzymes. Single-cell RNAseq transcriptomics (and to some extend proteomics) may provide information on the repertoire of expressed GTs and other relevant enzymes, and this may be useful for the engineering design considering the current knowledge of glycosylation pathways in cells (Fig. 2A). However, it is important to acknowledge that interpretation of such data still requires caution. Further advances are needed to be able to reliably predict glycosylation outcomes based on quantitative levels of enzymes in cells, and the predicted roles of GTs in the different glycosylation pathways and biosynthetic steps need validation.
      Targeting biosynthetic steps in glycosylation that are controlled nonredundantly by a single unique GT generally results in predictable global outcomes. For example, KO of FUT8 that solely directs transfer of α1-6 fucose to the innermost GlcNAc residue of the chitobiose core of N-glycans is sufficient to eliminate this glycosylation feature on N-glycoproteins (
      • Yamane-Ohnuki N.
      • Kinoshita S.
      • Inoue-Urakubo M.
      • Kusunoki M.
      • Iida S.
      • Nakano R.
      • Wakitani M.
      • Niwa R.
      • Sakurada M.
      • Uchida K.
      • Shitara K.
      • Satoh M.
      Establishment of FUT8 knockout Chinese hamster ovary cells: An ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.
      ,
      • Malphettes L.
      • Freyvert Y.
      • Chang J.
      • Liu P.Q.
      • Chan E.
      • Miller J.C.
      • Zhou Z.
      • Nguyen T.
      • Tsai C.
      • Snowden A.W.
      • Collingwood T.N.
      • Gregory P.D.
      • Cost G.J.
      Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies.
      ,
      • Wang X.
      • Inoue S.
      • Gu J.
      • Miyoshi E.
      • Noda K.
      • Li W.
      • Mizuno-Horikawa Y.
      • Nakano M.
      • Asahi M.
      • Takahashi M.
      • Uozumi N.
      • Ihara S.
      • Lee S.H.
      • Ikeda Y.
      • Yamaguchi Y.
      • et al.
      Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.
      ). Many steps in the initiation and immediate core extension of glycosylation pathways are controlled by nonredundant enzymes (Fig. 2A), and this is used to dissect roles of elaborated glycans on different types of glycoconjugates by targeting the earliest committed biosynthetic steps in core extension. Thus, KO of the Glc-Cer β4-galactosyltransferase B4GALT5(6) gene(s) eliminates synthesis of elaborated glycolipids, KO of the α3-mannosyl-glycoprotein β2-GlcNAc-transferase MGAT1 gene eliminates elaboration of N-glycans, and KO of the core1 synthase C1GALT1 gene or the private chaperone COSMC eliminates elaboration of the most common types of O-glycan (
      • Narimatsu Y.
      • Joshi H.J.
      • Nason R.
      • Van Coillie J.
      • Karlsson R.
      • Sun L.
      • Ye Z.
      • Chen Y.H.
      • Schjoldager K.T.
      • Steentoft C.
      • Furukawa S.
      • Bensing B.A.
      • Sullam P.M.
      • Thompson A.J.
      • Paulson J.C.
      • et al.
      An atlas of human glycosylation pathways enables display of the human glycome by gene engineered cells.
      ,
      • Dabelsteen S.
      • Pallesen E.M.H.
      • Marinova I.N.
      • Nielsen M.I.
      • Adamopoulou M.
      • Romer T.B.
      • Levann A.
      • Andersen M.M.
      • Ye Z.
      • Thein D.
      • Bennett E.P.
      • Bull C.
      • Moons S.J.
      • Boltje T.
      • Clausen H.
      • et al.
      Essential functions of glycans in human epithelia dissected by a CRISPR-cas9-engineered human organotypic skin model.
      ,
      • Stolfa G.
      • Mondal N.
      • Zhu Y.
      • Yu X.
      • Buffone Jr., A.
      • Neelamegham S.
      Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and glycosphingolipids to human leukocyte-endothelium adhesion.
      ,
      • Steentoft C.
      • Vakhrushev S.Y.
      • Vester-Christensen M.B.
      • Schjoldager K.T.
      • Kong Y.
      • Bennett E.P.
      • Mandel U.
      • Wandall H.
      • Levery S.B.
      • Clausen H.
      Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.
      ,
      • Byrne G.
      • O'Rourke S.M.
      • Alexander D.L.
      • Yu B.
      • Doran R.C.
      • Wright M.
      • Chen Q.
      • Azadi P.
      • Berman P.W.
      CRISPR/Cas9 gene editing for the creation of an MGAT1-deficient CHO cell line to control HIV-1 vaccine glycosylation.
      ,
      • Stewart S.E.
      • Menzies S.A.
      • Popa S.J.
      • Savinykh N.
      • Petrunkina Harrison A.
      • Lehner P.J.
      • Moreau K.
      A genome-wide CRISPR screen reconciles the role of N-linked glycosylation in galectin-3 transport to the cell surface.
      ). Rational engineering may lead to discoveries and challenge the current understanding of genetic and biosynthetic regulation of glycosylation pathways. For example, when the—at the time—known genes controlling protein O-mannosylation (POMT1/2) were KO in mammalian cells followed by analysis of the O-Man glycoproteome, two new O-mannosylation pathways directed by previously unknown enzymes were discovered (
      • Larsen I.S.B.
      • Narimatsu Y.
      • Joshi H.J.
      • Yang Z.
      • Harrison O.J.
      • Brasch J.
      • Shapiro L.
      • Honig B.
      • Vakhrushev S.Y.
      • Clausen H.
      • Halim A.
      Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2.
      ,
      • Larsen I.S.B.
      • Narimatsu Y.
      • Joshi H.J.
      • Siukstaite L.
      • Harrison O.J.
      • Brasch J.
      • Goodman K.M.
      • Hansen L.
      • Shapiro L.
      • Honig B.
      • Vakhrushev S.Y.
      • Clausen H.
      • Halim A.
      Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins.
      ). Another example of glycoengineering leading to discoveries involved the use of forward genetic screening to identify the two enzymes directing the Galβ1-3GalNAcβ1-4 branch attached to the first Man residue of the glycosylphosphatidylinositol (GPI)-anchor (
      • Hirata T.
      • Mishra S.K.
      • Nakamura S.
      • Saito K.
      • Motooka D.
      • Takada Y.
      • Kanzawa N.
      • Murakami Y.
      • Maeda Y.
      • Fujita M.
      • Yamaguchi Y.
      • Kinoshita T.
      Identification of a Golgi GPI-N-acetylgalactosamine transferase with tandem transmembrane regions in the catalytic domain.
      ). The post-GPI attachment to proteins GalNAc-transferase 4 (PGAP4) predicted to attach the first β4GalNAc was validated by KO of the PGAP4 gene, and a genome-wide CRISPR/Cas screen identified the β3-galactosyltransferase 4 (B3GALT4) as the enzyme extending the β4GalNAc (
      • Wang Y.
      • Maeda Y.
      • Liu Y.S.
      • Takada Y.
      • Ninomiya A.
      • Hirata T.
      • Fujita M.
      • Murakami Y.
      • Kinoshita T.
      Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation.
      ). B3GALT4 also functions as the GM1 glycolipid synthase (
      • Amado M.
      • Almeida R.
      • Carneiro F.
      • Levery S.B.
      • Holmes E.H.
      • Nomoto M.
      • Hollingsworth M.A.
      • Hassan H.
      • Schwientek T.
      • Nielsen P.A.
      • Bennett E.P.
      • Clausen H.
      A family of human beta3-galactosyltransferases. Characterization of four members of a UDP-galactose:beta-N-acetyl-glucosamine/beta-nacetyl-galactosamine beta-1,3-galactosyltransferase family.
      ,
      • Miyazaki H.
      • Fukumoto S.
      • Okada M.
      • Hasegawa T.
      • Furukawa K.
      Expression cloning of rat cDNA encoding UDP-galactose:GD2 beta1,3-galactosyltransferase that determines the expression of GD1b/GM1/GA1.
      ). A cell model was first engineered by KO of GPI transamidase component PIG-S gene (PIGS), encoding a subunit of the GPI-Tase that transfers GPI to proteins, to block protein transfer of GPIs and enable screening for loss of galactosylation by an antibody detecting the Galβ1-3GalNAcβ epitope on free GPIs. Interestingly, in the process of dissecting the putative dual role of B3GALT4, it was discovered that UGCG that initiates glycolipid biosynthesis is required for B3GALT4 functions in both glycolipid and GPI biosynthesis. These studies thus not only uncovered an unexpected cross talk between two different glycosylation pathways, but also led to discovery of interaction between UGCG and B3GALT4. The Ribitol β4-xylosyltransferase (TMEM5) required for biosynthesis of the core O-mannosyl matriglycan of dystroglycan was originally identified in a HAP1 screen and validated by KO showing that the Xylβ1-4Rbo5P structure was disrupted (
      • Manya H.
      • Yamaguchi Y.
      • Kanagawa M.
      • Kobayashi K.
      • Tajiri M.
      • Akasaka-Manya K.
      • Kawakami H.
      • Mizuno M.
      • Wada Y.
      • Toda T.
      • Endo T.
      The muscular dystrophy gene TMEM5 encodes a ribitol beta1,4-xylosyltransferase required for the functional glycosylation of dystroglycan.
      ).
      A “brutal” variant of rational glycoengineering is to target genes that serve glycosylation in global ways such as in synthesis and transport of nucleotide sugar donors, e.g., as demonstrated already with the CHO Lec mutants (
      • Patnaik S.K.
      • Stanley P.
      Lectin-resistant CHO glycosylation mutants.
      ). KO of SLC35A1 results in loss of all types of sialylation (
      • Stanley P.
      • Sudo T.
      • Carver J.P.
      Differential involvement of cell surface sialic acid residues in wheat germ agglutinin binding to parental and wheat germ agglutinin-resistant Chinese hamster ovary cells.
      ,
      • Riemersma M.
      • Sandrock J.
      • Boltje T.J.
      • Bull C.
      • Heise T.
      • Ashikov A.
      • Adema G.J.
      • van Bokhoven H.
      • Lefeber D.J.
      Disease mutations in CMP-sialic acid transporter SLC35A1 result in abnormal alpha-dystroglycan O-mannosylation, independent from sialic acid.
      ). The original discovery that loss of the UDP-Glc/GlcNAc C4-epimerase (GALE) in the CHO ldld cell model resulted in deficiencies of UDP-Gal/GalNAc and impaired glycosylation of N- and O-glycoproteins, a defect that can be reversed by exogenous addition of Gal and/or GalNAc sugars (
      • Kingsley D.M.
      • Kozarsky K.F.
      • Hobbie L.
      • Krieger M.
      Reversible defects in O-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant.
      ,
      • Kozarsky K.
      • Kingsley D.
      • Krieger M.
      Use of a mutant cell line to study the kinetics and function of O-linked glycosylation of low density lipoprotein receptors.
      ), is now being replicated in other cells by targeted KO of GALE/GALK1/2 to install the unique ability to regulate glycosylation by exogenous addition of sugars (
      • Termini J.M.
      • Silver Z.A.
      • Connor B.
      • Antonopoulos A.
      • Haslam S.M.
      • Dell A.
      • Desrosiers R.C.
      HEK293T cell lines defective for O-linked glycosylation.
      ,
      • Boyce M.
      • Carrico I.S.
      • Ganguli A.S.
      • Yu S.H.
      • Hangauer M.J.
      • Hubbard S.C.
      • Kohler J.J.
      • Bertozzi C.R.
      Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway.
      ). Similarly, KO of the isoprenoid synthase domain-containing protein ISPD required for synthesis of CDP-Ribitol blocks synthesis of the matriglycan on α-dystroglycan (
      • Gerin I.
      • Ury B.
      • Breloy I.
      • Bouchet-Seraphin C.
      • Bolsee J.
      • Halbout M.
      • Graff J.
      • Vertommen D.
      • Muccioli G.G.
      • Seta N.
      • Cuisset J.M.
      • Dabaj I.
      • Quijano-Roy S.
      • Grahn A.
      • Van Schaftingen E.
      • et al.
      ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto alpha-dystroglycan.
      ,
      • Riemersma M.
      • Froese D.S.
      • van Tol W.
      • Engelke U.F.
      • Kopec J.
      • van Scherpenzeel M.
      • Ashikov A.
      • Krojer T.
      • von Delft F.
      • Tessari M.
      • Buczkowska A.
      • Swiezewska E.
      • Jae L.T.
      • Brummelkamp T.R.
      • Manya H.
      • et al.
      Human ISPD is a cytidyltransferase required for dystroglycan O-mannosylation.
      ).
      Targeting biosynthetic steps that are covered by partial redundancy from isoenzymes remains a challenge for predicting outcomes of rational glycoengineering. However, targeting isoenzymes also present unique opportunities to uncover their nonredundant functions. For isoenzymes that function in pathway specific steps, such as the many polypeptide GalNAc-transferases (GALNTs), selective KO/KI of individual isoenzyme genes (GALNT1-20) in cell models was very useful to dissect non-redundant functions using differential O-glycoproteomics (
      • Schjoldager K.T.
      • Joshi H.J.
      • Kong Y.
      • Goth C.K.
      • King S.L.
      • Wandall H.H.
      • Bennett E.P.
      • Vakhrushev S.Y.
      • Clausen H.
      Deconstruction of O-glycosylation--GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.
      ,
      • Schjoldager K.T.
      • Vakhrushev S.Y.
      • Kong Y.
      • Steentoft C.
      • Nudelman A.S.
      • Pedersen N.B.
      • Wandall H.H.
      • Mandel U.
      • Bennett E.P.
      • Levery S.B.
      • Clausen H.
      Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells.
      ,
      • Bagdonaite I.
      • Pallesen E.M.
      • Ye Z.
      • Vakhrushev S.Y.
      • Marinova I.N.
      • Nielsen M.I.
      • Kramer S.H.
      • Pedersen S.F.
      • Joshi H.J.
      • Bennett E.P.
      • Dabelsteen S.
      • Wandall H.H.
      O-glycan initiation directs distinct biological pathways and controls epithelial differentiation.
      ). This strategy provides deeper and more unbiased insights into substrates for GALNTs compared with in vitro enzyme assays with short peptides and has revealed important isoform-specific targets such as the O-glycosylation of the ligand-binding region of the low density lipoprotein receptor-related receptors directed exclusively by GALNT11 (
      • Kong Y.
      • Joshi H.J.
      • Schjoldager K.T.
      • Madsen T.D.
      • Gerken T.A.
      • Vester-Christensen M.B.
      • Wandall H.H.
      • Bennett E.P.
      • Levery S.B.
      • Vakhrushev S.Y.
      • Clausen H.
      Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis.
      ,
      • Wang S.
      • Mao Y.
      • Narimatsu Y.
      • Ye Z.
      • Tian W.
      • Goth C.K.
      • Lira-Navarrete E.
      • Pedersen N.B.
      • Benito-Vicente A.
      • Martin C.
      • Uribe K.B.
      • Hurtado-Guerrero R.
      • Christoffersen C.
      • Seidah N.G.
      • Nielsen R.
      • et al.
      Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions.
      ,
      • Tian E.
      • Wang S.
      • Zhang L.
      • Zhang Y.
      • Malicdan M.C.
      • Mao Y.
      • Christoffersen C.
      • Tabak L.A.
      • Schjoldager K.T.
      • Ten Hagen K.G.
      Galnt11 regulates kidney function by glycosylating the endocytosis receptor megalin to modulate ligand binding.
      ). A particularly useful extension of this strategy is to install inducible expression of GT isoenzymes to explore their functions in regulating glycosylation. Thus, engineering cells with KO of an endogenous GALNT and reinstallation of the same isoenzyme under stringent inducible expression by KI enabled the remarkable observation that increasing levels of the GALNT isoenzyme produce tight regulation of nonredundant substrates without interference or change to the majority of redundant substrates (
      • Hintze J.
      • Ye Z.
      • Narimatsu Y.
      • Madsen T.D.
      • Joshi H.J.
      • Goth C.K.
      • Linstedt A.
      • Bachert C.
      • Mandel U.
      • Bennett E.P.
      • Vakhrushev S.Y.
      • Schjoldager K.T.
      Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines.
      ). KO screen of GALNTs was used to show that GALNT1 plays an essential role in the glycosylation of the mucin domain of the Ebola surface glycoprotein and the function of this in cell detachment and hemorrhaging (
      • Simon E.J.
      • Linstedt A.D.
      Site-specific glycosylation of Ebola virus glycoprotein by human polypeptide GalNAc-transferase 1 induces cell adhesion defects.
      ). Engineering GALNT genes in a human keratinocyte skin model showed that distinct isoforms serve unique functions in epithelial formation and differentiation (
      • Dabelsteen S.
      • Pallesen E.M.H.
      • Marinova I.N.
      • Nielsen M.I.
      • Adamopoulou M.
      • Romer T.B.
      • Levann A.
      • Andersen M.M.
      • Ye Z.
      • Thein D.
      • Bennett E.P.
      • Bull C.
      • Moons S.J.
      • Boltje T.
      • Clausen H.
      • et al.
      Essential functions of glycans in human epithelia dissected by a CRISPR-cas9-engineered human organotypic skin model.
      ). Similarly, a characteristic de novo expression of the GALNT6 isoenzyme in colon cancer was investigated by engineering GALNTs in a colon cancer cell line, and GALNT6 was selectively shown to affect differentiation and growth (
      • Lavrsen K.
      • Dabelsteen S.
      • Vakhrushev S.Y.
      • Levann A.M.R.
      • Haue A.D.
      • Dylander A.
      • Mandel U.
      • Hansen L.
      • Frodin M.
      • Bennett E.P.
      • Wandall H.H.
      De novo expression of human polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6) in colon adenocarcinoma inhibits the differentiation of colonic epithelium.
      ). The unique functions of several other GALNT isoenzymes were also explored in KO studies (
      • Khetarpal S.A.
      • Schjoldager K.T.
      • Christoffersen C.
      • Raghavan A.
      • Edmondson A.C.
      • Reutter H.M.
      • Ahmed B.
      • Ouazzani R.
      • Peloso G.M.
      • Vitali C.
      • Zhao W.
      • Somasundara A.V.
      • Millar J.S.
      • Park Y.
      • Fernando G.
      • et al.
      Loss of function of GALNT2 lowers high-density lipoproteins in humans, nonhuman primates, and rodents.
      ,
      • Lackman J.J.
      • Goth C.K.
      • Halim A.
      • Vakhrushev S.Y.
      • Clausen H.
      • Petaja-Repo U.E.
      Site-specific O-glycosylation of N-terminal serine residues by polypeptide GalNAc-transferase 2 modulates human delta-opioid receptor turnover at the plasma membrane.
      ,
      • Goth C.K.
      • Halim A.
      • Khetarpal S.A.
      • Rader D.J.
      • Clausen H.
      • Schjoldager K.T.
      A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation.
      ,
      • Goth C.K.
      • Tuhkanen H.E.
      • Khan H.
      • Lackman J.J.
      • Wang S.
      • Narimatsu Y.
      • Hansen L.H.
      • Overall C.M.
      • Clausen H.
      • Schjoldager K.T.
      • Petaja-Repo U.E.
      Site-specific O-glycosylation by polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) co-regulates beta1-adrenergic receptor N-terminal cleavage.
      ,
      • de Las Rivas M.
      • Paul Daniel E.J.
      • Narimatsu Y.
      • Companon I.
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      • et al.
      Molecular basis for fibroblast growth factor 23 O-glycosylation by GalNAc-T3.
      ,
      • Xu Y.
      • Pang W.
      • Lu J.
      • Shan A.
      • Zhang Y.
      Polypeptide N-acetylgalactosaminyltransferase 13 contributes to neurogenesis via stabilizing the mucin-type O-glycoprotein podoplanin.
      ,
      • Al Rifai O.
      • Julien C.
      • Lacombe J.
      • Faubert D.
      • Lira-Navarrete E.
      • Narimatsu Y.
      • Clausen H.
      • Ferron M.
      The half-life of the bone-derived hormone osteocalcin is regulated through O-glycosylation in mice, but not in humans.
      ). Moreover, glycoengineering is being used with approaches for metabolic labeling and tagging of glycosylation (
      • Agatemor C.
      • Buettner M.J.
      • Ariss R.
      • Muthiah K.
      • Saeui C.T.
      • Yarema K.J.
      Exploiting metabolic glycoengineering to advance healthcare.
      ,
      • Moons S.J.
      • Adema G.J.
      • Derks M.T.
      • Boltje T.J.
      • Bull C.
      Sialic acid glycoengineering using N-acetylmannosamine and sialic acid analogs.
      ,
      • Prescher J.A.
      • Bertozzi C.R.
      Chemical technologies for probing glycans.
      ,
      • Belardi B.
      • Bertozzi C.R.
      Chemical lectinology: Tools for probing the ligands and dynamics of mammalian lectins in vivo.
      ,
      • Hong S.
      • Yu C.
      • Wang P.
      • Shi Y.
      • Cao W.
      • Cheng B.
      • Chapla D.G.
      • Ma Y.
      • Li J.
      • Rodrigues E.
      • Narimatsu Y.
      • Yates 3rd, J.R.
      • Chen X.
      • Clausen H.
      • Moremen K.W.
      • et al.
      Glycoengineering of NK cells with glycan ligands of CD22 and selectins for B-cell lymphoma therapy.
      ), including the azido sugars for click chemistry introduced by the Bertozzi group (
      • Prescher J.A.
      • Dube D.H.
      • Bertozzi C.R.
      Chemical remodelling of cell surfaces in living animals.
      ,
      • Saxon E.
      • Bertozzi C.R.
      Cell surface engineering by a modified Staudinger reaction.
      ,
      • Laughlin S.T.
      • Baskin J.M.
      • Amacher S.L.
      • Bertozzi C.R.
      In vivo imaging of membrane-associated glycans in developing zebrafish.
      ). Glycoengineered cells allow for screening and validation of selective binding and labeling probes in live cells as exemplified by the bump-and-hole strategy employed by Schumann and colleagues (
      • Choi J.
      • Wagner L.J.S.
      • Timmermans S.
      • Malaker S.A.
      • Schumann B.
      • Gray M.A.
      • Debets M.F.
      • Takashima M.
      • Gehring J.
      • Bertozzi C.R.
      Engineering orthogonal polypeptide GalNAc-transferase and UDP-sugar pairs.
      ), in which modified UDP-GalNAc donor substrates (bumped) and GALNTs engineered to selectively accommodate these by an enlarged active site (hole) are used to detect isoform-specific functions in transfected cells (
      • Schumann B.
      • Malaker S.A.
      • Wisnovsky S.P.
      • Debets M.F.
      • Agbay A.J.
      • Fernandez D.
      • Wagner L.J.S.
      • Lin L.
      • Li Z.
      • Choi J.
      • Fox D.M.
      • Peh J.
      • Gray M.A.
      • Pedram K.
      • Kohler J.J.
      • et al.
      Bump-and-hole engineering identifies specific substrates of glycosyltransferases in living cells.
      ,
      • Debets M.F.
      • Tastan O.Y.
      • Wisnovsky S.P.
      • Malaker S.A.
      • Angelis N.
      • Moeckl L.K.R.
      • Choi J.
      • Flynn H.
      • Wagner L.J.S.
      • Bineva-Todd G.
      • Antonopoulos A.
      • Cioce A.
      • Browne W.M.
      • Li Z.
      • Briggs D.C.
      • et al.
      Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation.
      ,
      • Cioce A.
      • Malaker S.A.
      • Schumann B.
      Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools.
      ).
      In N-glycosylation the functions of the two dolichyl-diphosphooligosaccharide protein GT STT3A and STT3B subunits of the heteromeric OST complex were analyzed by proteomics studies of HEK293 cells with KO of either subunit, demonstrating distinct modification sites for the STT3A/B subunits (
      • Cherepanova N.A.
      • Venev S.V.
      • Leszyk J.D.
      • Shaffer S.A.
      • Gilmore R.
      Quantitative glycoproteomics reveals new classes of STT3A- and STT3B-dependent N-glycosylation sites.
      ,
      • Cherepanova N.A.
      • Gilmore R.
      Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation.
      ). Similarly, KO targeting was used to demonstrate that both FKTN and FKRP are Ribitol 5-phosphate transferases (
      • Kanagawa M.
      • Kobayashi K.
      • Tajiri M.
      • Manya H.
      • Kuga A.
      • Yamaguchi Y.
      • Akasaka-Manya K.
      • Furukawa J.I.
      • Mizuno M.
      • Kawakami H.
      • Shinohara Y.
      • Wada Y.
      • Endo T.
      • Toda T.
      Identification of a post-translational modification with ribitol-phosphate and its defect in muscular dystrophy.
      ), that both procollagen galactosyltransferase 1 (COLGALT1) and 2 (COLGALT2) function in galactosylation of hydroxylysine (HYL) in collagens (
      • Baumann S.
      • Hennet T.
      Collagen accumulation in osteosarcoma cells lacking GLT25D1 collagen galactosyltransferase.
      ,
      • Hennet T.
      Collagen glycosylation.
      ), that both protein O-glucosyltransferase 2 (POGLUT2) and 3 (POGLUT3) serve the same Notch EGF11 repeat sequence (C3-XNTXGSFX-C4) different from POGLUT1 (
      • Takeuchi H.
      • Schneider M.
      • Williamson D.B.
      • Ito A.
      • Takeuchi M.
      • Handford P.A.
      • Haltiwanger R.S.
      Two novel protein O-glucosyltransferases that modify sites distinct from POGLUT1 and affect Notch trafficking and signaling.
      ), and that the substrate specificities of some of the four DPY19L1-4 C-mannosyltransferases may differ with respect to Trp substrate sites in consecutive glycosylation site motifs (e.g., the WXXWXXWXXC sequence in thrombospondin repeats found in the netrin receptor UNC5A) (
      • Shcherbakova A.
      • Tiemann B.
      • Buettner F.F.
      • Bakker H.
      Distinct C-mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3.
      ).
      However, most isoenzymes function in glycosylation pathway nonspecific steps with considerable cross talk between pathways, and these isoenzymes direct terminal structural features that determine many of the biological interactions with, e.g., GBPs (Fig. 2A). This includes the enzymes assembling the elongation and branching of LacNAc disaccharides (Galβ1-3/4GlcNAcβ1-R) and LacDiNAc (GalNAcβ1-4GlcNAcβ1-R) termini and the capping steps by the many sialyltransferase and fucosyltransferase isoenzymes (
      • Harduin-Lepers A.
      • Vallejo-Ruiz V.
      • Krzewinski-Recchi M.A.
      • Samyn-Petit B.
      • Julien S.
      • Delannoy P.
      The human sialyltransferase family.
      ,
      • de Las Rivas M.
      • Lira-Navarrete E.
      • Gerken T.A.
      • Hurtado-Guerrero R.
      Polypeptide GalNAc-Ts: From redundancy to specificity.
      ,
      • de Vries T.
      • Knegtel R.M.
      • Holmes E.H.
      • Macher B.A.
      Fucosyltransferases: Structure/function studies.
      ,
      • Paulson J.C.
      • Rademacher C.
      Glycan terminator.
      ,
      • Becker D.J.
      • Lowe J.B.
      Fucose: Biosynthesis and biological function in mammals.
      ,
      • Tsuji S.
      Molecular cloning and functional analysis of sialyltransferases.
      ,
      • Oriol R.
      • Mollicone R.
      • Cailleau A.
      • Balanzino L.
      • Breton C.
      Divergent evolution of fucosyltransferase genes from vertebrates, invertebrates, and bacteria.
      ,
      • Togayachi A.
      • Sato T.
      • Narimatsu H.
      Comprehensive enzymatic characterization of glycosyltransferases with a beta3GT or beta4GT motif.
      ). Since these glycan structures are found widely on N-glycans, O-glycans, and glycolipids, glycoengineering may affect different types of glycoconjugates and complicate analysis and assignments of functions. Redundant functions of the B4GALTs (B4GALT1-4) in galactosylation of N-glycans have been shown by combinatorial KO in CHO cells (
      • Yang Z.
      • Wang S.
      • Halim A.
      • Schulz M.A.
      • Frodin M.
      • Rahman S.H.
      • Vester-Christensen M.B.
      • Behrens C.
      • Kristensen C.
      • Vakhru