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| Open AccessA doxycycline- and light-inducible Cre recombinase mouse model for optogenetic genome editing
Achieving spatial control of gene expression is important. Here the authors report an optimised photoactivatable Cre recombinase system, doxycycline- and light-inducible Cre recombinase (DiLiCre), and generate a DiLiCre mouse line which they use for mutagenesis in vivo and positional cell-tracing.
- Miguel Vizoso
- , Colin E. J. Pritchard
- & Jacco van Rheenen
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Article
| Open AccessTransient inhibition of p53 enhances prime editing and cytosine base-editing efficiencies in human pluripotent stem cells
Li et al. report that co-delivering a dominant negative fragment of p53 (p53DD) greatly enhances precise editing efficiencies for prime editing and cytosine base editing in human pluripotent stem cells, without compromising the genome-wide safety.
- Mu Li
- , Aaron Zhong
- & Ting Zhou
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Article
| Open AccessGlobal mapping of GalNAc-T isoform-specificities and O-glycosylation site-occupancy in a tissue-forming human cell line
Information about O-glycosylation site regulation and occupancy in the human proteome is limited. Here, the authors identify GalNAc transferase-specific glycan sites in human keratinocytes and describe their occupancy.
- Mathias I. Nielsen
- , Noortje de Haan
- & Hans H. Wandall
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Article
| Open AccessA synthetic transcription platform for programmable gene expression in mammalian cells
Precise and scalable regulation of gene expression in mammalian cells is challenging. Here, the authors created a highly tunable CRISPR-based synthetic transcription system for programmable control of mammalian gene expression and cellular activity.
- William C. W. Chen
- , Leonid Gaidukov
- & Timothy K. Lu
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Article
| Open AccessMarker-free co-selection for successive rounds of prime editing in human cells
Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. Here the authors develop a co-selection strategy to facilitate prime editing in human cells and provide design principles to prevent the formation of undesired editing byproducts at the target site.
- Sébastien Levesque
- , Diana Mayorga
- & Yannick Doyon
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Article
| Open AccessLive imaging and conditional disruption of native PCP activity using endogenously tagged zebrafish sfGFP-Vangl2
Planar cell polarity (PCP) is critical for tissue-wide coordination and successful development. Here Jussila et al. generate a GFP-Vangl2 fusion for live imaging and discover a surprising directionality to the intercellular propagation of cell polarity, and ultimately link PCP defects with idiopathic scoliosis.
- Maria Jussila
- , Curtis W. Boswell
- & Brian Ciruna
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Article
| Open AccessSystematic profiling of conditional degron tag technologies for target validation studies
Conditional Degron Tags are a valuable tool to validate and study novel therapeutic targets. Here, the authors compared 5 orthogonal tags across 16 unique proteins and provide a panel of vectors for users to systematically screen the tags with their own protein of interest.
- Daniel P. Bondeson
- , Zachary Mullin-Bernstein
- & Alessandra Ianari
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Article
| Open AccessGeneration of a CRISPR activation mouse that enables modelling of aggressive lymphoma and interrogation of venetoclax resistance
Modelling of aggressive lymphomas, such as double hit lymphoma, has been challenging. Here the authors engineer a CRISPR activation mouse to enable the generation of these aggressive lymphomas and identify the pro-survival BCL-2 protein A1 as a venetoclax resistance factor.
- Yexuan Deng
- , Sarah T. Diepstraten
- & Marco J. Herold
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Article
| Open AccessMassively targeted evaluation of therapeutic CRISPR off-targets in cells
Thorough evaluation of CRISPR RNA-guided nucleases off-targets in cells is required for advancing gene therapies. Here the authors report SURRO-seq for the simultaneous investigation of thousands of off-target sites for therapeutic RNA-guided nucleases in cells.
- Xiaoguang Pan
- , Kunli Qu
- & Yonglun Luo
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Article
| Open AccessNucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes to enhance RNAi stability in plants
Long hairpin RNA (hpRNA) transgenes are the most widely used RNAi technology in plants, but are potentially subject to self-induced transcriptional silencing. Here, the authors show nucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes in Arabidopsis and tobacco.
- Daai Zhang
- , Chengcheng Zhong
- & Ming-Bo Wang
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Article
| Open AccessPeptide fusion improves prime editing efficiency
Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. Here the authors present PepSEq, a high-throughput method for screening a large library of peptides that influence prime editing efficiency.
- Minja Velimirovic
- , Larissa C. Zanetti
- & Richard I. Sherwood
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Article
| Open AccessDisease modeling by efficient genome editing using a near PAM-less base editor in vivo
Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. Here the authors describe a near PAM-less base editor and its application in zebrafish to efficiently create disease models harbouring specific point mutations.
- Marion Rosello
- , Malo Serafini
- & Filippo Del Bene
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Article
| Open AccessA multifunctional system for genome editing and large-scale interspecies gene transfer
The need for diverse chromosomal modifications in biotechnology, synthetic biology and basic research requires the development of new technologies. Here the authors present CRISPR SWAPnDROP, which extends the limits of genome editing to large-scale in-vivo DNA transfer between bacterial species.
- Marc Teufel
- , Carlo A. Klein
- & Patrick Sobetzko
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Article
| Open AccessCCN1 interacts with integrins to regulate intestinal stem cell proliferation and differentiation
Intestinal stem cells contribute to homeostasis through a balance between self-renewal and differentiation. Here the authors show that CCN1 is an intestinal stem cell niche factor that activates integrin αvβ3/αvβ5 signaling to regulate proliferation and differentiation through distinct downstream pathways.
- Jong Hoon Won
- , Jacob S. Choi
- & Joon-Il Jun
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Article
| Open AccessTransient expression of an adenine base editor corrects the Hutchinson-Gilford progeria syndrome mutation and improves the skin phenotype in mice
Base editing to treat diseases is progressing but tissue delivery and progenitor cells correction are challenging. Here, the authors show sustained effects and propagation of mutation-corrected progenitors by transient adenine base editor expression, improving the skin phenotype of HGPS mice.
- Daniel Whisenant
- , Kayeong Lim
- & Maria Eriksson
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Article
| Open AccessA framework to efficiently describe and share reproducible DNA materials and construction protocols
DNA constructs and their annotated sequence maps have been rapidly accumulating with the advancement of DNA cloning, synthesis, and assembly methods. Here the authors introduce QUEEN, a framework to describe and share DNA materials and construction protocols.
- Hideto Mori
- & Nozomu Yachie
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Article
| Open AccessAn in vivo gene amplification system for high level expression in Saccharomyces cerevisiae
Gene dosage-based expression upregulation suffers from instability and random gene integration. Here, the authors report HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification, and demonstrate its applications in protein and biochemical production in yeast.
- Bingyin Peng
- , Lygie Esquirol
- & Claudia E. Vickers
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Article
| Open AccessGenome editing in animals with minimal PAM CRISPR-Cas9 enzymes
PAM requirement is a constraint for genome editing but this has been circumvented by engineered Cas9 nucleases as SpG and SpRY recognizing minimal PAM sequences. Here, the authors validate and optimize SpG and SpRY in vivo expanding the targeting landscape in animals.
- Jeremy Vicencio
- , Carlos Sánchez-Bolaños
- & Miguel A. Moreno-Mateos
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Article
| Open AccessTargeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies
Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. Here the authors report the development of the double tap - double tap implements secondary gRNAs which target Cas9 to common indel sequences and provides a second chance at HDR.
- Zsolt Bodai
- , Alena L. Bishop
- & Alexis C. Komor
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| Open AccessEngineering artificial photosynthetic life-forms through endosymbiosis
The endosymbiotic theory posits that chloroplasts in eukaryotes arise from bacterial endosymbionts. Here, the authors engineer the yeast/cyanobacteria chimeras and show that the engineered cyanobacteria perform chloroplast-like functions to support the growth of yeast cells under photosynthetic conditions.
- Jason E. Cournoyer
- , Sarah D. Altman
- & Angad P. Mehta
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Article
| Open AccessCRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus
Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Here the authors present a CRISPR-based approach for labeling the genome at multiple nonrepetitive loci in living cells and to image chromatin loops in the presence and absence of cohesin.
- Patricia A. Clow
- , Menghan Du
- & Albert W. Cheng
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Article
| Open AccessEfficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci
Trans-chromosomic (Tc) mice have helped the development of therapeutic antibodies, but chromosome instability limits its application. Here the authors develop a new line of Tc mice with full human Ig heavy and kappa loci integrated into the mouse artificial chromosome for stable passage, and confirm efficient generation of B cell responses and specific antibodies.
- Hiroyuki Satofuka
- , Satoshi Abe
- & Yasuhiro Kazuki
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Article
| Open AccessHighly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure
Prime editors can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here the authors introduce same-sense mutations into pegRNAs to increase base-editing efficiency and the pegRNA secondary structure was altered to increase indel-editing efficiency.
- Xiaosa Li
- , Lina Zhou
- & Jia Chen
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Article
| Open AccessPhage peptides mediate precision base editing with focused targeting window
Base editors are genome engineering tools that can generate nucleotide substitutions without introducing double-stranded breaks. Here the authors show that a phage-derived peptidyl CRISPR inhibitor can be employed to modulate the activity and targeting scope of CRISPR base editor for precision base editing applications.
- Kun Jia
- , Yan-ru Cui
- & Jia Liu
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Article
| Open AccessFrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity
Gene editing tools have tremendous potential for biomedical and basic research. Here the authors report a Cas9 from Faecalibaculum rodentium (FrCas9) that achieves efficient and specific gene editing in human cells with a NNTA palindrome PAMs for targeting optimal sites at TATA-boxes to enhance CRISPRa/i screening.
- Zifeng Cui
- , Rui Tian
- & Zheng Hu
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Article
| Open AccessMutation-specific reporter for optimization and enrichment of prime editing
While prime editing is a promising technique, some genomic sites remain difficult to edit. Here the authors present fluoPEER, fluorescent prime editing and enrichment reporter, to rank the efficiency of pegRNAs and prime editor variants.
- I. F. Schene
- , I. P. Joore
- & S. A. Fuchs
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Article
| Open AccessChemically Induced Chromosomal Interaction (CICI) method to study chromosome dynamics and its biological roles
Methods to selectively manipulate specific long-distance chromosomal interactions are limited. Here the authors develop a method called Chemically Induced Chromosomal Interaction (CICI) to engineer interactions and demonstrate that 3D conformation plays a causal role in establishing donor DNA preference during DNA repair.
- Manyu Du
- , Fan Zou
- & Lu Bai
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Article
| Open AccessA pan-CRISPR analysis of mammalian cell specificity identifies ultra-compact sgRNA subsets for genome-scale experiments
Context specificity confounds genetic analysis and prevents reproducible genome engineering. Here, the authors report a pan-CRISPR analysis of specificity in mammalian cells and identify ultra-compact sgRNA subsets for genome-scale screens.
- Boyang Zhao
- , Yiyun Rao
- & Justin R. Pritchard
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Article
| Open AccessImproved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage
Some DNA sequences are refractory to CRISPR-Cas9 cleavage, partially due to gRNA misfolding. Here the authors engineer gRNAs to prevent misfolding and further enhanced their stability by chemical modifications allowing robust genome editing regardless of target sequence.
- Stephan Riesenberg
- , Nelly Helmbrecht
- & Svante Pääbo
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Article
| Open AccessGenome-wide detection of CRISPR editing in vivo using GUIDE-tag
In vivo assessment of nuclease off-target activity has primarily been indirect or through ChIP-based detection of double-strand break DNA repair factors, which can be cumbersome. Here, the authors show that GUIDE-tag, enables one-step off-target genome editing analysis in mouse liver and lung.
- Shun-Qing Liang
- , Pengpeng Liu
- & Wen Xue
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Article
| Open AccessExpression of the transcription factor PU.1 induces the generation of microglia-like cells in human cortical organoids
The study of human microglia function in health and disease is limited by the availability of sound models. Here, the authors develop a method to generate functional microglia in human cortical organoids and investigate the role of human microglia during amyloid beta1-42- induced inflammation.
- Bilal Cakir
- , Yoshiaki Tanaka
- & In-Hyun Park
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Article
| Open AccessNuclear and mitochondrial DNA editing in human cells with zinc finger deaminases
Base editing in nuclear DNA and mitochondrial DNA (mtDNA) is broadly useful for biomedical research, medicine, and biotechnology. Here the authors present zinc finger deaminases which catalyze targeted C-to-T base conversions without inducing unwanted indels in human cells.
- Kayeong Lim
- , Sung-Ik Cho
- & Jin-Soo Kim
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Article
| Open AccessReversing insecticide resistance with allelic-drive in Drosophila melanogaster
Insecticide resistance (IR) poses a major global health challenge. Here, the authors generate common IR mutations in laboratory Drosophila strains and use a CRISPR-based allelic-drive to replace an IR allele with a susceptible wild-type counterpart, providing a potent new tool for vector control.
- Bhagyashree Kaduskar
- , Raja Babu Singh Kushwah
- & Ethan Bier
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| Open AccessExploiting a Y chromosome-linked Cas9 for sex selection and gene drive
CRISPR-based engineering can be used to bias sex ratios. Here the authors develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome and functionally characterize the utility of this strain for both sex selection and gene drive.
- Stephanie Gamez
- , Duverney Chaverra-Rodriguez
- & Omar S. Akbari
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| Open AccessCRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes
In areas such as animal research and agriculture a single sex is often required in abundance, leading to wasted resources and ethical considerations. Here the authors develop a CRISPR/Cas9 mediated synthetic lethal system that enables the production of single sex offspring that can be repurposed for use in multiple organisms.
- Charlotte Douglas
- , Valdone Maciulyte
- & James M. A. Turner
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Article
| Open AccessFind and cut-and-transfer (FiCAT) mammalian genome engineering
Mammalian genome engineering has advanced tremendously over the last decade, however there is still a need for robust gene writing with size scaling capacity. Here the authors present Find Cut-and-Transfer (FiCAT) technology to delivery large targeted payload insertion in cell lines and in vivo in mouse models.
- Maria Pallarès-Masmitjà
- , Dimitrije Ivančić
- & Marc Güell
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| Open AccessSelf-organization of human dorsal-ventral forebrain structures by light induced SHH
Organizing centers act to pattern surrounding tissues during embryogenesis through the secretion of morphogens. Here the authors model human organizers using light stimulus to geometrically confine SHH expression in differentiating hESCs, generating spatially resolved proximal distal patterns.
- Riccardo De Santis
- , Fred Etoc
- & Ali H. Brivanlou
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| Open AccessHomecoming: rewinding the reductive evolution of the chloroplast genome for increasing crop yields
Developing more productive and sustainable crops will be essential to achieving food security in coming decades. A core process in plant evolution has been the transfer of chloroplast-encoded genes to the nuclear genome. We propose reverting this process as a new approach to improve plant disease resistance and photosynthesis in future crops.
- Briardo Llorente
- , María Eugenia Segretin
- & Nicolás E. Blanco
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Article
| Open AccessAsymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc
Morphogens disperse to pattern tissues and control their growth during development, allowing for the specification of multiple fates across space. Here the authors block dispersal of a morphogen Dpp (BMP2/4) and show that the requirement for Dpp dispersal is much lower than previously thought.
- Shinya Matsuda
- , Jonas V. Schaefer
- & Markus Affolter
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Article
| Open AccessBEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors
Base editors allow for precision engineering of the genome. Here, the authors present BEAR, a plasmid-based fluorescence assay for the measurement of CBE and ABE activity, to reveal the mechanism underlying their differences and to increase the yield of edited cells with reduced indel background.
- András Tálas
- , Dorottya A. Simon
- & Ervin Welker
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Article
| Open AccessAugmented CO2 tolerance by expressing a single H+-pump enables microalgal valorization of industrial flue gas
Microalgae used for CO2 removal in an industrial exhaust gas stream usually has low CO2 tolerance. Here, the authors increase CO2 tolerance by expressing a single H + -pump and enable microalgal valorization of industrial flue gas.
- Hong Il Choi
- , Sung-Won Hwang
- & Sang Jun Sim
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Article
| Open AccessDisulfide-compatible phage-assisted continuous evolution in the periplasmic space
The directed evolution of antibodies yields important tools for research and therapy. Here the authors develop a periplasmic phage-assisted continuous evolution platform for improvement of protein-protein interactions in the disulfidecompatible E. coli periplasm.
- Mary S. Morrison
- , Tina Wang
- & David R. Liu
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Article
| Open AccessA standardized genome architecture for bacterial synthetic biology (SEGA)
Genome engineering is challenging compared to plasmid DNA manipulation. Here the authors create a simple methodology called SEGA that enables genome engineering by combining DNA and bacterial cells followed by identification of recombinant clones by a change in colour when grown on agar plates.
- Carolyn N. Bayer
- , Maja Rennig
- & Morten H. H. Nørholm
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Article
| Open AccessIn vivo inducible reverse genetics in patients’ tumors to identify individual therapeutic targets
Preclinical molecular models are useful that mimic a patient´s response to targeted therapy. Here, the authors establish an in vivo inducible RNAi-mediated gene silencing system in patient-derived xenograft models of acute leukemia to identify individual vulnerabilities and therapeutic targets.
- Michela Carlet
- , Kerstin Völse
- & Irmela Jeremias
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Article
| Open AccessGeneration of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain
While prime editing is a promising technology, PE2 systems often have low efficiency. Here the authors fuse a Rad51 DNA-binding domain to create hyPE2 with improved editing efficiency.
- Myungjae Song
- , Jung Min Lim
- & Hyongbum Henry Kim
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Article
| Open AccessA CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses
Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Here the authors generate mutant clonal intestinal organoids for 19 host genes previously implicated in coronavirus biology and identify the cell surface protease TMPRSS2 as a potential therapeutic target.
- Joep Beumer
- , Maarten H. Geurts
- & Hans Clevers
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Article
| Open AccessDynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice
The in vivo identification of proteins secreted from a specific cell type or tissue remains challenging. Here, the authors develop a proximity labeling-based method to selectively label secreted proteins and combine it with proteomics to identify liver secretory proteins in mouse plasma.
- Kwang-eun Kim
- , Isaac Park
- & Jae Myoung Suh
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Article
| Open AccessEnhanced regulation of prokaryotic gene expression by a eukaryotic transcriptional activator
Expanded toolkits for prokaryotic synthetic biology can enhance the dynamic range of gene expression. Here the authors move the eukaryotic transcription factor QF into E. coli and integrate it into genetic devices.
- I. Cody MacDonald
- , Travis R. Seamons
- & Tara L. Deans
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Article
| Open AccessDisplay of the human mucinome with defined O-glycans by gene engineered cells
Mucins play critical roles in maintaining the human microbiome, with their O-glycosylated tandem repeats (TRs) providing important cues for microbiota. Here, the authors develop a cellular platform for producing TRs with defined O-glycan structures to dissect the functions of TR O-glycosylation.
- Rebecca Nason
- , Christian Büll
- & Yoshiki Narimatsu